| Background: Rheumatoid arthritis(RA)is a slow developing autoimmune disease,the etiology of which is not completely clear.It affects about 1.0% of the world’s total population.It is also a disease that causes damage to our people’s labor force.Relevant research data show that Th17 cell and Treg cell are very important in the nosogenesis of RA.Tripterygium hypoglaucum hutch(THH)belongs to the Tripterygium wilfordii of celastraceae.In recent years,many studies have shown that THH can improve the inflammatory symptoms of rheumatoid arthritis and relieve the pain of patients,so whether THH can inhibit inflammation by regulating the balance of Th17 cell and Treg cell has not been reported.Objective: In this experiment,the Collagen-induced arthritis(CIA)model was established to detect the changes of Th17 cells,Treg cells and their transcription factors and cytokines in CIA mice after THH treatment,so as to explore the immunological mechanism of THH.Methods: 40 healthy male C57BL/6 mice and 40 healthy female C57BL/6 mice were selected as reserve mice,and 24 mice were randomly selected as normal control group with half males and half females.Female mice were assigned to A1 group and male mice to A2 group.The remaining 56 mice are used for the preparation of CIA mice model.The successfully mice(arthritis index score ≥ 4)were randomly split into 4 groups: respectively is the CIA model group B1 of female,male mice CIA model group B2,female THH treatment group C1 and male mice THH treatment group C2,12 mice in each group and the mice in the group were given intragastric administration for 20 days.Mice in C1 and C2 treatment groups were given intragastric administration of THH,while mice in A1 and A2 normal control groups,B1 and B2 model groups were given the equal volume of normal saline instead of THH.During this period,the activity volume,body weight,thickness of ankle joint,foot joint and arthritis index of mice were dynamically monitored.After intragastric administration,the synovial membrane of mice joints was stained with HE to observe the changes of synovial inflammation.Real-time quantitative PCR(RT-q PCR)was used to detect the expressions of ROR γ T mRNA and Foxp3 mRNA in spleens of mice,as well as the expressions of IL-17 AmRNA and TGF-βmRNA in the joints of mice,and the IL-17,IL-10 and TGF-β in serum of mice were determined by enzyme-linked immunosorbent assay(ELISA).Results: Among the 56 model mice,the arthritis index score of 52 mice met the criteria(arthritis index score ≥ 4),and the success rate of modeling was 92.8%.After 20 days of intragastric administration,the joints of female and male mice in group A were not swollen,the arthritis index(AI)was less than 1,the activity of mice was normal and the body weight increased slightly,and the joint swelling of both hindlimbs in group B was obvious,and the AI value of group B1 was higher than that of group B2 on the22 days after initial immunization,the difference was statistically significant.The activity of both female and male mice decreased,and the body weight of the two groups decreased slightly after the second immunization,but there was no significantly different compared with group A.The degree of redness and swelling of ankle joint and paw of group C decreased with the prolongation of treatment time,and the AI value decreased significantly,but the AI value of group C1 was higher than that of group C2 since the 22 th day of the first immunization.Later period of continuous medication,the activity and body weight of mice increased,and there was no significantly different compared with group B.HE staining results: monolayer of flat synovial cell was seen in the synovial layer of mice in group A under arthroscopy,and there were no inflammatory cell infiltration and neovascularization.The continuity of articular cartilage was good,and no bone destruction was observed.In group B,the proliferation of cells in synovial layer and new blood vessels were observed under ankle arthroscopy,and inflammatory cells in subsynovial layer and poor bone integrity were observed.Under ankle arthroscopy in group C,the number of subsynovial inflammatory cells and the number of subsynovial cells were reduced,and the bone erosion was improved.There was no significant difference between males and females in ABC group.RT-q PCR results: the relative expression of ROR γ t mRNA in spleen in group B1(8.9900 ±0.9100)was higher than that in group C1(3.7200 ±0.2800)(p < 0.0001),and that in group B2(7.3400 ±0.7900)was higher than that in group C2(3.6200 ±0.2600)(p < 0.0001).There was no significantly different between B1 and B2,C1 and C2.The relative expression of Foxp3 mRNA in spleen in B1 group(0.4300±0.0700)was lower than that in C1 group(0.7800 ±0.0300)(p<0.05),and that in B2 group(0.4800 ±0.0900)was lower than that in C2 group(0.7500 ±0.0600)(p < 0.0l).There was significantly different between B1 and B2,C1 and C2 groups.The relative expression of IL-17 AmRNA in B1 group(1.8690 ±0.3657)was higher than that in C1 group(0.2621 ±0.0462)(p < 0.0001),B2 group(0.9625 ±0.1309)was higher than that in C2 group(0.2001 ±0.0825)(p <0.0001),B1 group was higher than B2 group(p < 0.001),there was no significantly different between C1 group and C2 group.The relative expression of TGF-β mRNA in group B1(0.2637 ±0.0501)was lower than that in group C1(0.5637 ±0.0499)(p < 0.0001),and that in group B2(0.3000 ±0.0499)was lower than that in group C2(0.6027 ±0.0540)(p < 0.0001).There was no significantly different between B1 and B2,C1 and C2.ELISA results: the concentration of serum IL-17 A in B1 group(6.2110 ±0.3250pg/m L)was higher than that in C1 group(4.0020 ±0.3150pg/m L)(p < 0.0001)and A1 group(2.5030 ±0.1350pg/m L)(p < 0.0001).(4.9660 ±0.6020pg/m L)in group B2 was higher than that in group C2(3.2990 ±0.2350pg/m L)(p < 0.01)and higher than that in group A2(2.0030 ±0.1350pg/m L)(p < 0.0001),that in group B1 was higher than that in group B2(p < 0.05),the difference between C1 group and C2 group has no statistical meaning.The concentration of IL-10 in group B1(515.2740 ±25.9950pg/m L)was lower than that in group C1(750.2578±33.9649pg/m L)(p < 0.0001)and that in group A1(915.9155 ±36.2526pg/m L)(p < 0.0001).The level of B2 group(506.7460 ±36.5840pg/m L)was lower than that of C2 group(709.6345 ±31.6479pg/m L)(p < 0.0001)and A2 group(845.5674 ±16.2843pg/m L)(p < 0.0001),there was no statistical meaning in B1 and B2,C1 and C2.The concentration of TGF-β in B1 group(2280.3750±146.3090pg/m L)was lower than that in C1 group(2896.2640 ±97.7390pg/m L)(p < 0.01)and A1 group(4248.4220 ±135.8070pg/m L)(p < 0.0001).group B2(2668.9640 ±132.0750pg/m L)was lower than C2(3208.6060 ±179.4130pg/m L)(p<0.05)and A2(4278.8530 ±126.7820pg/m L)(p < 0.0001),and there was significantly different between B1 and B2,C1 and C2.Conclusions: THH can alleviate the articular inflammation of CIA mice by down-regulating the expression of RORγt mRNA and up-regulating the expression of Foxp3 mRNA in the spleen of the peripheral immune organ,as well as decreasing the levels of IL-17 and increasing the levels of IL-10 and TGF-β in the microenvironment.It is suggested that THH can have therapeutic effect on RA patients by affecting the number of Th17 cells and Treg cells in RA microenvironment. |
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