Effect Of Sinomenine On The HPA Axis, Corticosterone Receptor In PBMC And The Expression Of CRH And ACTH In Synovium Of Rats With Adjuvant Arthritis

Objective: Rheumatoid arthritis (RA) is a kind of autoimmune disease characterized by chronic and symmetrical multijoint inflammation, which result in joint malformation even lead to sever disability. The major damaged-target is synovial membrane. The cause of RA is still unclear. Recently, the role of hypothalamic-pituitary-adrenal (HPA) axis has triggered scientists'interests following the development of the role of Neuroendocrine-Immunity network in RA. HPA axis is important to suppress inflammatory reaction and regulate immune function. Glucocorticoid receptor (GCR) is not only the mediator of immune signals, but also the effective tool of immune regulation. The change of the binding capacity of GCR in peripheral blood mononuclear cells (PBMC) has inner relationship with the development of RA. The non-specific anti-inflammatory effect of sinomenine may be shown by the way of stimulating HPA axis and increasing secretion of adrenal cortical hormone. However, there are few reports about the clinical and experimental research on the HPA axis mechanism of sinomenine in the treatment of RA. We, therefore, established the adjuvant-induced arthritis (AA) rats model and made a further study on the effect of sinomenine on joint inflammation, concentrations of corticotropin release hormone (CRH), adrenocorticotrophic hormone (ACTH), and corticosterone in plasma, expression of CRH and ACTH in synovium, and expression of GCR in PBMC, in order to explore the role of HPA axis in the pathogenesy of RA and the therapeutic effect of sinomenine.Methods: (1) 125 male Wistar rats were randomly and evenly divided into five groups,Ⅰ: normal group;Ⅱ: model group;Ⅲ: model group with low dosage of sinomenine treatment (15mg/kg·d);Ⅳ: model group with medium dosage of sinomenine treatment (30mg/kg·d);Ⅴ: model group with high dosage of sinomenine treatment (60mg/kg·d). The AA rats of groupⅡ,Ⅲ,ⅣandⅤwere established by intradermal injection of complete Freund's adjuvant(CFA). The success rate was evaluated by arthritis index (AI). Each group was divided into 5 subgroups according to the time point (7d, 14d, 28d, 35d and 42d) after the establishment of AA model. (2) From the 14th day after the establishment of AA model, rats of groupⅢ,ⅣandⅤwere injected with sinomenine of different dosages. (3) The paw thickness was measured and AI was evaluated at different time point. (4) The concentrations of CRH, ACTH and corticosterone in plasma were detected by radioimmunoassay (RIA) at different time point. (5) The expression of GCR in PBMC was measured by radio-ligand binding assay using 3H-dexamethansone as the ligand. (6) Immunohistochemistry was used to detect the expression of CRH and ACTH in synovium and intensity was analyzed by computerized image system. (7) Histopathology of ankle joint was observed and evaluated using histopathology integral. (8) The data were inputted into a computer and were analyzed by statistical package for the social science (SPSS) 14.0.Results: (1)AI: On different time after the establishment of AA model, AI of groupⅡwas significantly higher than that of groupⅠ(P<0.01) and it reached peak value on the 28th day. On the 35th~42nd days, AI of groupⅣandⅤwas lower than that of groupⅡ(P<0.05), but AI of groupⅢwas similar with that of groupⅡ(P>0.05). There was no statistic difference among groupⅢ,ⅣandⅤ. (2)paw thickness: From the 14th day to the 42nd day after the establishment of AA model, the paw thickness of groupⅡ~Ⅴwas significantly higher than that of groupⅠ(P<0.01) and the peak time was the 21st ~28th days. On the 28th day, the paw thickness of groupⅤwas lower than that of groupⅡ(P<0.05), and the time was earlier than groupⅣfor 14 days. There was no statistic difference between groupⅢand groupⅡon different time. On the 28th day, the paw thickness of groupⅤwas lower than that of groupⅢ(P<0.05), and the time was earlier than groupⅣfor 7 days. On the 35th day, the paw thickness of groupⅤwas lower than that of groupⅣ(P<0.05). (3) Concentration of CRH in plasma: CRH of groupⅡ~Ⅴwas lower than that of groupⅠon the 35th day (P<0.01). On the 42nd day, although CRH of groupⅢ~Ⅴincreased compared with that on the 35th day(P<0.05), CRH of groupⅡ~Ⅴwas still lower than that of groupⅠ. CRH of groupⅣ~Ⅴwas higher than that of groupⅡon the 35th day (P<0.05), and CRH of groupⅢwas higher than that of groupⅡon the 42nd day (P<0.01). It implied that high dosage and medium dosage of sinomenine could increase the expression of CRH in plasma on the 35th day, and the time was earlier than low dosage for 7 days. On the 35th ~42nd days, CRH of groupⅣwas higher than that of groupⅢ, and CRH of groupⅤwas higher than that of groupⅣ. It implied that high dosage showed the strongest effect, and low dosage showed the weakest effect. (4) Concentration of ACTH in plasma: ACTH of groupⅡ~Ⅴwas higher than that of groupⅠon the 14th day (P<0.01) and the peak time was the 28th day. Although ACTH of groupⅢ~Ⅴdecreased in the following time(P<0.05), it was still higher than that of groupⅠuntil the 42nd day. ACTH of groupⅤwas lower than that of groupⅡon the 35th day (P<0.05), and ACTH of groupⅣwas lower than that of groupⅡon the 42nd day (P<0.01). It implied that high dosage of sinomenine could reduce the expression of ACTH in plasma on the 35th day, and the time was earlier than medium dosage for 7 days. Low dosage of sinomenine had no therapeutic effect during the experiment. On the 35th day, ACTH of groupⅤwas lower than that of groupⅢ. On the 42nd day, ACTH of groupⅣwas lower than that of groupⅢ, and ACTH of groupⅤwas lower than that of groupⅣ. It implied that high dosage of sinomenine showed the effect the most rapidly and the most evidently. (5) Concentration of corticosterone in plasma: Corticosterone of groupⅡ~Ⅴwas lower than that of groupⅠon the 14th day (P<0.01) and the trough time was the 28th day. Although corticosterone of groupⅣ~Ⅴincreased in the following time(P<0.05), it was still lower than that of groupⅠuntil the 42nd day(P<0.01). Corticosterone of groupⅣ~Ⅴwas higher than that of groupⅡon the 35th~42nd days (P<0.05), but there was no statistic difference between groupⅢand groupⅡon different time. It implied that high dosage and medium dosage of sinomenine could increase the expression of corticosterone in plasma on the 35th~42nd days, but low dosage had no effect until the experiment finished. On the 35th day, corticosterone of groupⅤwas higher than that of groupⅢandⅣ(P<0.05), and there was no statistic difference between groupⅢand groupⅣ. On the 42nd day, corticosterone of groupⅣwas higher than that of groupⅢ, and corticosterone of groupⅤwas higher than that of groupⅣ(P<0.05). It implied that high dosage of sinomenine showed the effect more evidently than medium dosage. (6) The expression of GCR in PBMC: The expression of GCR of groupⅡwas lower than that of groupⅠ(P<0.01) . After being treated with high dosage of sinomenine for 28 days, the expression of GCR increased compared with that of groupⅡ(P<0.05), but it was still lower than that of groupⅠ(P<0.05). It implied that sinomenine could increase the expression of GCR in PBMC. (7) The expression of CRH and ACTH in synovium: The expression of CRH and ACTH in synovium of groupⅡ~Ⅴwere higher than those of groupⅠ(P<0.01) on different time, especially for groupⅡ. The peak time was the 28th day. Sinomenine could reduce the expression of CRH and ACTH in synovium. High dosage of sinomenine showed that effect on the 28th day, medium dosage on the 35th day, and low dosage on the 42nd day. On the 28th day, The expression of CRH and ACTH of groupⅤwere lower than those of groupⅢandⅣ. On the 35th~42nd days, The expression of CRH and ACTH of groupⅣwere lower than those of groupⅢ, and the expression of CRH and ACTH of groupⅤwere lower than those of groupⅣ(P<0.01). It implied that high dosage of sinomenine showed the effect the most rapidly and the most evidently. (8)Histopathological integral of joint: Integral of groupⅡwas higher than that of groupⅠon different time, and the peak time was the 28th~42nd days. Sinomenine of different dosages could reduce integral and the high dose had the strongest effect. On the 35th~42nd days, integral of groupⅤwas lower than that of groupⅢandⅣ. (9) The correlation analysis: Paw thickness was positively related with AI, the concentration of ACTH in plasma, and the expression of CRH and ACTH in synovium (P<0.01). Paw thickness was negtively related with the concentrations of CRH and corticosterone in plasma (P<0.01). The concentration of ACTH in plasma was positively related with the expression of CRH and ACTH in synovium. The expression of CRH in synovium was positively related with the expression of ACTH in synovium (P<0.01).Conclusion: (1) AI was positively related with paw thickness. They could both reflect the swelling of joints, but the latter was preciser and more objective. (2) After the establishment of AA model, the concentrations of CRH and corticosterone in plasma decreased while the concentration of ACTH increased, which implied hypothalamic and adrenal defects. Sinomenine could adjust the function of the HPA axis, but it still couldn't reach the normal level. The effect of sinomenine was due to the dosage and time. The high dose showed the effect most rapidly and evidently. (3) After the establishment of AA model, the expression of GCR in PBMC decreased and the high dosage of sinomenine could increase it, which implied the low expression of GCR in the pathological process of AA and sinomenine's therapeutic effect. (4) After the establishment of AA model, the expression of CRH and ACTH in synovium increased, higher than the normal level, and sinomenine could reduce them. Its therapeutic effect was related to the dosage and time. (5) Sinomenine could effectively reduce the histopathological integral and suppress the pathological change of joints.