| ObjectiveTo Observe the effect of Bushen-Qiangdu-Zhilv Decoction on immune inflammation in Ankylosing Spondylitis animal model,and the influence on the IL-23/Th17 axis in model.MethodsChoose to use the BALB/c mice for zeroth days,Twentieth days,fortieth day tail subcutaneous injection of bovine cartilage proteoglycan induced arthritis model(PGIA),as the animal model of ankylosing spondylitis.These mice were randomly divided into four groups as control group,model group,Bushen-Qiangdu-Zhilv Decoction group and celecoxib group,10 mices in each group.The control group was normal BALB/c mice,and the other groups were operated by PGIA.On day 61-90,control group and model group were received daily gavage of saline,Bushen-Qiangdu-Zhilv Decoction group were administration with clinical equivalent dose of water extract of Bushen-Qiangdu-Zhilv Decoction,celecoxib group were administration with clinical equivalent dose of celecoxib solution by gavage once a day.At the end of the experiment,the mice were sacrificed.The main experimental indicators including arthritis score,small animal in vivo imaging observation,joint swelling of paw;spleen index and thymus index(TI)measurement;extraction of spleen total lymphocyte proliferation by CCK-8 cell measurement.Analysis the proportion of Th17 cells by flow cytometry.Collection of serum,enzyme linked immunosorbent assay(ELISA)method for measuring TNF-alpha,interleukin IL-17,the expression level of IL-23 protein.ELISA method for IL-23 and IL-17 ileum tissue protein measurement;using HE staining method,immunohistochemical staining and safranin solid green 0 staining of the sacroiliac joint,lumbar vertebral joint or tail for histopathological observation.Results1.Compared with the control group,BALB/c mice by bovine proteoglycans after repeated immunization,spontaneous occurrence of hind paw swelling of joint and arthritis scores were significantly increased(P<0.001);SI and TI increased significantly(P<0.05);the expression level of serum TNF-a increased significantly(P<0.001);expression the serum IL-17 increased significantly(P<0.001);the expression level of IL-23 was significantly higher than that of control group(P<0.001);the total splenic lymphocytes in vitro after 24 hours of activity significantly decreased compared to the control group(P<0.001);Th17 cells accounted for the total proportion of lymphocytes increased;there was no significant difference between IL-17 and IL-23 protein expression in the intestinal wall and the control group(P>0.05);histopathological staining showed that PGIA mice of sacroiliac joint cartilage metaplasia increased gradually,sacroiliac joint fusion of intervertebral disc,inflammatory cell infiltration,fibrous ring structure and damage Angiogenesis,the destruction of the coccygeal vertebrae,proved the successful construction of PGIA mouse model.The IL-17 positive cells in the PGIA mice were significantly higher than those in the control group(P<0.001)and IL-23(P<0.01).2.Compared with the model group,Bushen-Qiangdu-Zhilv Decoction group joint swelling score decreased significantly(P<0.05);SI and TI index decreased;Water extract of Bushen-Qiangdu-Zhilv Decoction cultured 24 hours in vitro,the total lymphocyte activity were significantly increased(P<0.01),prompting Bushen-Qiangdu-Zhilv water extract can promote the proliferation of mouse spleen lymphocytes total PGIA;Bushen-Qiangdu-Zhilv Decoction group serum TNF-expression level was significantly decreased compared with the model group(P<0.01);Th17 cells accounted for the total proportion of lymphocytes compared to the model group had no statistical significance.There was no significant difference between the intestinal the wall of IL-17,the expression level of IL-23 protein and the model group(P>0.05);HE staining found Bushen-Qiangdu-Zhilv Decoction group compared with model group joint bone destruction situation has improved,the edge joint surface more orderly and more;intervertebral disc tissue Complete,rare lymphocyte distribution.Safranin green staining found Bushen-Qiangdu-Zhilv Decoction group of sacroiliac joint intact intervertebral disc cartilage hyperplasia,reduce tail vertebra fusion phenomenon.Bushen-Qiangdu-Zhi lv Decoction group in the backbone of IL-17 positive cells were significantly higher than those in control group(P<0.001),the same IL-23 expression significantly(P<0.01),but the expression compared with the model group had no statistical significance(P>0.05).3.Compared with the model group,celecoxib group joint swelling score decreased,SI and TI index decreased;lymphocyte was cultured for 24 hours with 10nM celecoxib resolution,and the total lymphocyte activity was significantly increased compared with the model group(P<0.01),promting that celecoxib solution can promote the proliferation of spleen of PGIA mice total lymphocyte.It seems that expression of celecoxib group serum TNF-levels lower than those in the model group,but the difference was not statistically significant(P>0.05).Th17 cells accounted for the total proportion of lymphocytes compared to the model group was not statistically significant.There was no significant difference between the intestinal wall of IL-17,the expression level of IL-23 protein and the model group(P>0.05).HE staining was found in celecoxib group than the control group,the sacroiliac joint involvement situation has improved,but there is still a slight hyperplasia of articular cartilage,intervertebral disc structure slight damage.Safranin green staining found that celecoxib group of sacroiliac joint cartilage cells were intact,and a little angiogenesis.Celecoxib group in the backbone of IL-17 positive cells increased significantly compared with the control group(P<0.001),the same IL-23 expression significantly(P<0.05),but the expression compared with the model group had no statistical significance(P>0.05).4.Celecoxib as positive control drug in this study,the reduced joint swelling score,SI,TI,increasing the total lymphocyte activity,and Bushen-Qiangdu-Zhi lv Decoction were similar.Bushen-Qiangdu-Zhi lv Decoction group in improving inflammation in the body,maintain the sacroiliac joint and intervertebral disc structure integrity is slightly better than that of celecoxib group;but in the regulation of IL-23 and IL-17 on the backbone,celecoxib has a better downward trend,but the two compared with the model group were not statistically significant.Conclusion1.This study demonstrated that the PGIA mouse model was successfully constructed,and there was an imbalance of IL-23/Th17 axis in PGIA mice,which could be used to study the pathogenesis of AS and to screen the drug targeting IL-23/Thl7 axis.2.The data show that Bushen-Qiangdu-Zhilv Decoction intervention can improve PGIA mice inflammation,improve the sacroiliac joint and cartilage destruction,that Bushen-Qiangdu-Zhilv party intervention has a definite effect on PGIA mice model.3.Bushen-Qiangdu-Zhilv decoction has a clear effect on PGIA animal model in improving immune inflammation and bone dustruction,but may not by targeting the IL-23/Th17 axis function,presumably through other ways to further explore the effect,worthy of its mechanism. |
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