| Objective:To investigate the effects of different concentrations of Er miao San(traditional chinese medicine decoction)on the expression of NF-?Bp50 and ERK1/2 in collagen-induced arthritis(CIA)rats with rheumatoid arthritis(RA).Methods:60 female SPF grade Wistar were established as CIA model and intragastric infused by different concentrations of Er miao San.The CIA model rats were randomly divided into blank control,model group,and Er miao san group(divided into low,medium and high dose group respectively).Rat weight and joint scoring was recorded weekly.After 6-weeks intragastric infusion,X-ray examination and carotid blood collection was conducted under anesthesia,and then killed.Elisa detected changes in serum IL-6,TNF-? and IL-10 levels.Rat joints were taken to do HE pathology and to observe the pathological changes of rat joint tissues.qRT-PCR andWestern Blot was used to detect the expression of NF-?Bp50and ERK1/2 mRNA and their expression at protein level in the rats' joints.And their localization was performed by immunohistochemical techniques.Results:1.The CIA rat model was established successfully in the experiment.2.RA inflammation index,pathology and X-ray test results(1)There was no significant difference between the hind feet of the CIA rats after the first immunization for 7 days and that of the blank group.After 14 to 42 days of intensive immunization,it was found that all the hind legs of the rats except the blank group had obvious swelling.Compared with the model group,the swelling degree of the Er miao San group was relieved after the 14-42 d administration.(2)There was no significant difference in the weight among the CIA rats after 21 days modeling(P> 0.05).Compared with the blank control group,CIA rats' weight was significantly lower(P<0.01).The effect of Ermiao San on the weight of CIA rats was compared with that of the blank control group.The weight of the other groups decreased with time.There was no significant difference in body weight during the modeling period,and the body weight decreased significantly during the modeling period(P<0.05).Except the blank control group,the weight of each group did not increase with feeding time.It indicated that they were affected by the disease.At any time point,there was no significant difference in weight among the Ermiao San groups(P> 0.05).(3)The arthritis scores in other groups were significantly lower than those in the model group(P<0.05).The 14th-day rat arthritis score in experimental groups was significantly higher than that of the blank control group(P<0.05).The arthritis score in the model group increased and the arthritis score in the Er miao san group significantly decreased over time(P<0.05).The arthritis score of the middle-dose Er miao san group decreased more significantly(P<0.01).(4)Compared with the model group,the level of IL-6 and TNF-? of the blank control group was significantly lower(P <0.05),and the levels of IL-10 were significantly higher(P<0.05).Compared with the model group,the IL-6 and TNF-? level decreased and the level of IL-10 increased in the Er miaosan group.Compared with the low and medium dose Er miaosan group,IL-10 level of the model group increased significantly(P <0.05).The IL-6 and TNF-? level in the middle dose Er miaosan group significantly decreased compared with the model group(P<0.05).(5)The articular surface of rats in the blank control group was covered with a thin layer of transparent cartilage.Their joint capsule cavity was smooth and covered with a thin layer of synovial cells.In the model group,there is a certain degree of joint cavity deformation and joint cartilage destruction due to synovial cell proliferation,hyperemic interstitial edema and inflammatory cell infiltration.Compared with the blank control group,joint synovial cell proliferation,interstitial congestion and edema,and inflammatory cell infiltration occurred in the Er miao san group.Compared with the model group,the joint synovial hyperplasia and articular cartilage destruction were alleviated to a certain extent in the Er miao san group.The condition of the joints recovered significantly in the middle dose Er miao san group and normal cartilage tissue was visible clearly.(6)X-ray film showed that the joint surface of the blank control rats was smooth,the structure of the upper and lower joint surfaces was clear,the joints were intact,the boneswere not damaged,the joint space was normal,and the soft tissues were not swollen.Soft tissue swelling,fusion of articular surface,and narrow joint cavity even disappeared were found in the CIA rats.Their joint structure is not clear,there is serious bone absorption and dissolution phenomenon,and the joint has been basically damaged.Such X-ray film proved that the CIA rat model was successfully constructed.The difference of the X-ray images among low,middle and high dose Er miao san groups was not significant.In terms of the degree of swelling of soft tissue,the clarity of articular surface structure and space,and the degree of joint damage,Er miao San group showed improvement compared with the model group.Compared with the blank control group,above pathological lesions still exist3.NF-?Bp50,ERK1 and ERK2 mRNA and protein levels in rat joint tissues(1)Relative quantitative analysis of NF-?Bp50,ERK1,and ERK2 mRNA levels in joint tissues of rats in each group showed that NF-?Bp50,ERK1,and ERK2 mRNAs were expressed to different degrees in the joint tissues of rats varying from different groups.The expression levels of NF-?Bp50,ERK1 and ERK2 mRNA in the joints of CIA rats were significantly higher than those in other groups(P<0.01).The relative expressions of NF-?Bp50,ERK1 and ERK2 mRNA in the joint tissues of rats in the medium dose Er miao San group were extremely significantly lower than the expression levels in the joints of rats in other groups(P<0.01).(2)The expressions of NF-?Bp50 in the blank control group,the low-dose Er miao san group and the high-dose Er miao san group were significantly lower than those of the model group(P<0.05),and that of the medium-dose Er miao san group was significantly lower than the model group(P<0.05).The expressions of ERK1 / 2 protein in the blank control group and three Er miao san groups were significantly lower than those in the model group(P<0.01).4.Localization of NF-?Bp50,ERK1 and ERK2 in rat joint tissuesPositive signals were detected in synovial fibroblasts,endothelial cells,stromal cells,and macrophages of rat joint tissues in different groups.The expression of NF-?Bp50,ERK1 and ERK2 in the medium-dose Er miao san group was significantly lower than thatof the other groups.Experimental group(P<0.05).Such data showed that NF-?Bp50,ERK1 and ERK2 expressions were expressed in joint tissues of rats in every group and the medium dose Er miao san could significantly inhibited the expression of NF-?Bp50,ERK1 and ERK2.Conclusion:1.The CIA rat model was established successfully in the experiment;2.confirm the truth ofNF-?Bp50 and ERK1 / 2 are involved in the occurrence and development of RA;3.Er miao san can affect the disease progression of RA through NF-?Bp50 and ERK1 / 2.4.Different concentrations Er miao san could inhibit the expression of inflammatory factors,NF-?Bp50 and ERK1 / 2,especially at the concentration of 3.0 g/kg. |
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