Experimental Research On Intervention With ImmuneResponses Of Mice With Collagen-induced Arthritis ToT-cells By Huangqi Glycoprotein

ObjectiveAs an autoimmune disease of joints pathologically characterized by synovitis, rheumatoid arthritis (RA) is a chronic, systemic and destructive.As traditional Chinese medicine for tonifying Qi, Huangqi is effective for immunity regulation, anti-aging and anti-cancer, widely used for clinical practices.Huangqi glycoprotein (HQGP) is a glycoprotein of natural plants extracted from a traditional Chinese medicine known as astragalus membranaceus by water. Previous experiments have suggested that HQGP is effective for immunosuppression in vitro. However, ways for HQGP to intervene with immune responses of mice with collagen-induced arthritis (CIA) to T-cell abnormalities and its targets remain to be further discussed.In this experiment, models were built for mice with CIA to observe impacts of HQGP upon spleens and morphology of ankle joints in these mice. Expressions of key transcription factors in different T cell subsets and relevant cytokines were detected. In addition, relationships between imbalance of peripheral immune cells and RA were discussed, to further illustrate pathogenic mechanism of immune responses to T-cell abnormalities in RA and explore the significance of intervention therapy for this part, thereby laying an experimental foundation for clinically treating RA with traditional Chinese medicine.Methods1、The CIA mice model was established and evaluated through the arthritis index.2、Methods Flow cytometry was used to detect and analyze the proportion of Th1、Th2, Th17 and Treg cells subgroups in peripheral blood.3.HE was used to observe the pathological changes of the spleen and joint tissues in each group.4、The transcription factors expression of T-bet、GATA-3、ROR-γt and Foxp3 in spleen tissues were detected by Western blot.ResultsI. Preparation of Models for Mice with Bovine TypeⅡ Collagen-induced Arthritis (CIA) and Morphological Observations1. Scores on joint swelling of mice with CIAJoints of modeled mice were red and swollen on the 10th day after injection of adjuvant. Such joint inflammation was gradually aggravated, and mean score met standards for models on the 42nd day (AI>=4). In the mean time, mice appeared to be languid with a decrease in food intake and movement disorder.2. Changes to AI before and after treatment in each group of mice with CIAAfter treatment by HQGP and HC, AI declined in all groups of mice with CIA, which suggested that both HC and HQGP were effective for treating CIA, red and swollen joints were improved for mice with CIA, the results weren’t statistically significant.3. Damages of joint tissues in each group of mice with CIAPathological observations of an optic microscope shown that joint cavity was intact in the group of normal mice, where no inflammatory cells were infiltrated. Intact joint surface, narrow joint cavity and some damaged cartilages were seen in mice of the model group. Compared with the model group, joint structures of mice were relatively intact and joint cavities were widened in all other treated groups.4. Damages of spleens in each group of mice with CIAPathological observations indicated that compared with the normal group, film got thicker and inflammatory cells infiltrated in the model group, where infiltration of inflammatory cells was detected on walls of central arteries and inflammatory edema contributed to tissue looseness. Compared with the model group, infiltration of inflammatory cells was improved in all treated groups.II. Impacts of HQGP upon Th1/Th2 Subsets of Mice with CIA1. Detecting ratio of Thl and Th2 in peripheral blood of each group of mice by flow cytometryAccording to experimental results, ratio of Thl and Th2 in peripheral blood was higher in the model group of mice with CIA than that in the normal group, and the difference was statistically significant (p<0.05). After treatment, the ratio declined in HC group, low and middle-dose groups of HQGP compared with the model group, presenting statistically significant difference (p<0.05). It could be also discovered from the results that Thl decreased to a larger extent than Th2 in low and middle-dose groups of HQGP than the model group.2. Detecting expressions of T-bet and GATA-3 in splenic tissues of mice by Western blotThe experimental results suggested that, expressions of T-bet and GATA-3 were higher in the model group of mice with CIA than the normal group (p<0.05). Compared with the model group, these expressions were lowered in low and middle-dose groups of HQGP and HC group (p<0.05). Although they also decreased in high-dose group of HQGP, it wasn’t statistically significant. Additionally, expressions of T-bet declined more drastically than those of GATA-3 in separately comparing low-dose and middle-dose groups of HQGP with the model group.III. Impacts of HQGP upon Thl7/Treg Subsets of Mice with CIA1. Detecting ratio of Thl7 and Treg in peripheral blood of each group of mice by flow cytometryThe experimental results suggested that, compared with the normal group, the ratio ofTh17 increased in peripheral blood of the model group, but the ratio of Treg declined; the difference between these two groups was statistically significant (p<0.05). There presented a significant increase in the ratio of Th17/Treg with statistical significance (p<0.05). After treatment, the ratio of Thl7 decreased in low, middle and high-dose groups compared with the model group. The ratio of Treg increased in groups of HC and low-dose HQGP, between which the difference was statistically significant (p<0.05). The results of Treg weren’t statistically significant in middle and high-dose groups of HQGP. The ratio of Thl7/Treg declined in groups of HC, low, middle and high-dose HQGP, among which the differences were statistically significant (p<0.05).2. Detecting expressions of ROR-yt and Foxp3 in splenic tissues of mice by Western blotAccording to experimental results, there was an increase in expressions of ROR-yt and ratio of ROR-yt/Foxp3, but a decrease in expressions ofFoxp3 (p<0.01) in the model group of mice with CIA compared with the normal group, between which these results were statistically significant (p<0.05). The expressions of ROR-yt were lower in groups of HC, low, mid and high-dose HQGP, whereas the expressions of Foxp3 increased in the group of low-dose HQGP (p<0.01) compared with the model group. The results concerning Foxp3 weren’t statistically significant in groups of middle and high-dose HQGP. The ratio of ROR-γt/Foxp3 decreased in groups of HC, low, middle and high-dose HQGP, where the results were statistically significant (p<0.05).Conclusion(1) By observing scores of arthrositis indexes and morphological indexes of joints and spleens for mice with CIA, the results suggested duplication of models in these mice and lesions of splenic tissues in each group of mice with CIA were successful(2) HQGP can effectively treat mice with CIA in the model group, perhaps because it promotes recovery of functions in the body and improves inflammatory joints of mice with CIA for achieving its therapeutic effects by regulating the balance between pro-inflammatory and anti-inflammatory factors and correcting the imbalance between Th1/Th2 and Th17/Treg.