Effects Of Chiropractic Therapy On T-Lymphocyte Subsets And T-bet/GATA-3, IFN-?/IL-4 MRNA In Immunosuppressive Model Rats

Objective:The purpose of this study was to investigate the effect of chiropractic therapy on immune function of cyclophosphamide(CTX)-induced immunosuppressive model rats and its possible mechanism,so as to provide experimental basis for the immunomodulatory effect of chiropractic therapy.Methods:SPF SD rats aged 4-5 weeks were randomly divided into the modeling group and the non-modeling group.The rats in the modeling group were injected with CTX solution continuously for 4 days according to the concentration of 50mg/kg.The model was verified by observing the appearance behavior of rats and observing the changes of spleen structure under the microscope with HE staining.Criteria for successful modeling:behavioral changes such as depilation,repose tiredness,and changes in spleen structure occurred in rats.After confirming the success of modeling,rats were randomly divided into two groups,the non-modeling group was divided into the blank group and the blank chiropractic group,and the modeling group was divided into the model group and the model chiropractic group,6 rats in each group,a total of 24 rats.The blank group and the model group only simulated the grasping action,and the chiropractic intervention was carried out in the chiropractic group and the model chiropractic group.Knead ridge therapy specific operation method:knead ridge left hand wear linen gloves,with safety press fixed in the rat's head,at the same time by the right hand thumb in the stomach with food,and ring finger,three finger abdomen is relatively hard,back on experimental rats skin for the operation on either side of the spine to knead,arrogant rat long strong point starting until the da-zhui acupuncture point in the direction of the head,this is 1times.After the third kneading,the skin of the operative site was lifted once every three times.The ridge kneading operation was started on the 2nd day after the successful molding,once a day and 21 times a time.The daily intervention time of ridge kneading was fixed at15:00-17:00,and the operation was continued for 10 days.The chiropractic operation was completed by a fixed person,and the chiropractic time of each rat was about 3min.After the treatment,the samples were weighed,the body weight was recorded and the thymus index of the rats was calculated.The structure of spleen was observed by HE staining.Flow cytometry(FCM)was used to detect CD3~+,CD4~+and CD8~+lymphocytes for T cell subsets analysis.Real-time fluorescence quantitative PCR(qPCR)was used to detect the expression levels of the spleen transcription factors T-bet,GATA-3 mRNA,and cytokines IFN-?and IL-4 mRNA,and the T-bet/GATA-3 mRNA and IFN-?/IL-4 mRNA were calculated respectively.Experimental data were compared between groups using single-factor analysis and pairwise comparison of LSD.Weight analysis of rats before and after intervention was compared using paired sample t test.Results:1.HE staining:the submucosal parenchyma of the spleen of the blank group was clearly structured,and the structures of white pulp and red pulp were clear and the boundaries remained intact.Large round lymphatic nodules and closely arranged internal lymphocytes could be seen in the white pulp.The structure of spleen in blank chiropractic group was clear,and the structure of red and white pith was also clear and complete.In the CTX model group,the boundaries of white pulp and red pulp in the spleen were not clear.The number and area of white pulp decreased.White pulp structure destruction,mostly small blue block,and scattered in the red pulp;Note the increased number of follicles and connective tissue,but no significant lymph nodules.The spleen structure of the model chiropractic group was relatively intact and clear,the white and red pith were also clearly visible,the number of lymphatic nodules increased,but the area was small.2.Body weight:before the intervention,compared with the blank group,the body weight values of the model chiropractic group and the model group were significantly decreased,with a significant difference(P<0.01).After chiropractic intervention,the body weight of rats in the model chiropractic group was higher than that in the model group(P<0.05).The weight of rats in the blank chiropractic group was higher than that in the blank group(P<0.05).3.Thymus index:the thymus index value of the model group was lower than that of the blank group,with statistical difference(P<0.01).The thymus index of the model chiropractic group was significantly higher than that of the model group(P<0.01).4.FCM:the percentage of CD3~+,CD4~+and CD8~+T cells in peripheral blood of the model group was significantly lower than that of the blank group,with statistical significance(P<0.01).The percentage of CD3~+T and CD4~+T cells in the peripheral blood of the model chiropractic group was higher than that of the model group(P<0.05).The percentage of CD3~+,CD4~+and CD8~+T cells in blank chiropractic group was higher than that in blank chiropractic group(P<0.05).5.QPCR:compared with the blank group,the expression levels of t-bet,IFN-?,T-bet/GATA-3 and IFN-?/IL-4 mRNA in the spleen of the model group were significantly decreased,with statistically significant differences(P<0.01),and the expression levels of gata-3 mRNA were significantly increased,with statistically significant differences(P<0.01).Compared with the model group,the expression of GATA-3 mRNA in the spleen of the model chiropractic group decreased,while the expression of T-bet/GATA-3 and IFN-?/IL-4mRNA increased,showing statistical difference(P<0.05).There was no significant difference in T-bet,GATA-3,IFN-?,IL-4,T-bet/GATA-3,IFN-?/IL-4 mRNA expression(P>0.05).Conclusion:1.Chiropractic therapy can increase the body weight,thymus index and the percentage of T lymphocyte subsets in peripheral blood in total white blood cells of CTX immunosuppressive model rats.2.Chiropractic therapy can increase the weight of normal rats and the percentage of T lymphocyte subsets in the total number of white blood cells in peripheral blood.3.Chiropractic therapy can up-regulate the expression of T-bet/GATA-3 mRNA and IFN-?/IL-4 mRNA in the spleen of the immunosuppressive CTX model rats,thus promoting the balanced development of Th1 and Th2 cells.