| ObjectiveThis study is to reveal the mechanism of Yishen Juanbi pill ameliorating bone destruction in Collagen Induced Arthritis(CIA)rats,ovariectomized CIA rats as the animal model,by the EphB4/ephrinB2 signal pathway,and to provide further experimental evidence to treatment of postmenopausal rheumatoid arthritis from the perspective of reinforcing kidney.Methods104 female SD rats were selected,at the age of 10 months,which were randomly divided into 3 groups according to their weight,12 in blank control group,30 in sham operation group and 62 in OVX group.The rats in the ovary group had their bilateral ovaries removed.The sham operation group was cuting off the adipose tissue around ovaries.During the operation,3 rats died in the sham operation group,and 9 rats died in the ovary group.11 weeks after ovariectomy,vaginal smear was taken 1 times a day for 5 days.The estrous cycle of rats was observed,and rats without obvious estrous cycle were selected as ovariectomized rats.The ovariectomized rats(n=53)were divided into OVX group(n=12),ovariectomized CIA group(OVX+CIA,n=13),ovariectomized CIA+EV group(OVX+CIA+EV,n=14),and ovariectomized CIA+Yishen Juanbi pills group(OVX+CIA+YSJB,n=14).The sham operation rats(n=27)were divided into sham operation group(Sham,n=12)and sham operation CIA group(CIA,n=15).Method of making ovariectomized rats:The rats were anesthetized with intraperitoneal injection of 45mg/kg of pentobarbital sodium,and abdominal fixation was performed at the end of the rib and 1cm away from the lateral part of the spine.After disinfection with 75%alcohol and iodine tincture,the skin and back muscles were cut,and the white fat mass was gently pulled out from the incision with an eye forceps,and the ovaries were visible.The lower part of the ovariy was ligated with surgical suture,then the ovaries were removed and the incisions were sutured and applied with antiphlogistic powder.The others were removed with the same method.Method of making CIA rat model:Collagen type II solution(The concentration is 2mg/ml)was mixed with the same volume incomplete freund’s adjuvant by drop.The final concentration of type Ⅱ collagen was lmg/ml.Collagen type Ⅱ solution was sufficient emulsification in ice bath with homogenate,to non-proliferation of drops in water.The emulsified mixture was injected subcutaneously at the root of the tail at 0.2ml/only，that is,200g type Ⅱ collagen/only.After 7 days of the first immunization,the subcutaneous injection at the end of the tail was O.lml/only,that is,1OOg type Ⅱ collagen/only.Method of making ovariectomized CIA rat model:First,bilateral ovaries were excised.After 12 weeks,the model of ovariectomized CIA rats was made by immunization according to the CIA modeling method.Method of making sham operation model:The sham operation procedure was the same as the OVX rat model,but no ovariectomy was removed,only the bilateral adipose tissue around the ovary was removed.After 12 weeks,the CIA model was made according to the above methods.Gavage and water supply of rats in each group after 14 days of immunization:OVX+CIA+YSJB group were lavaged with concentration of 0.129g/ml YiShen JuanBi pill solution,delivery volume was 1ml/100 g,the dosage of 1.29g/kg body weight.The OVX+CIA+EV group was given a water solution of estradiol valerate with a concentration of 0.01 mg/ml,with a volume of lml/10Og and a dose of 0.1 Img/kg.It was given once a day for 28 days.The rats of Control group,Sham group,OVX group,CIA group and OVX+CIA group were filled with equal volume of pure water according to the upper method.1.5h after the last dose,the rats were intraperitoneally injected with pentobarbital sodium at the dose of 45mg/kg.After taking the blood,the rats were executed quickly,and the knee,ankle joint and hind paw were fixed in neutral formalin.The evaluation of joint pathological damage,the analysis of bone microstructure and the number or differentiation of joint osteoclast in the joint and expression of EphB4 and ephrinB2 protein were detected.Results1 Effect of Yishen Juanbi pill on body weight of ovariectomized CIA ratsAt the 7th and 14th days,the body weight of OVX+CIA+YSJB group was significantly higher than that in Control,Sham and CIA groups,but obviously lower than that in OVX group.There was no significant difference in weight between OVX+CIA+YSJB and OVX+CIA groups and OVX+CIA+EV group.At the 21th and 28th days,The weight of OVX+CIA+YSJB group was significantly higher than that of CIA group,but significantly lower than OVX group.Compared with OVX+CIA+EV group,there was no significant difference between OVX+CIA+YSJB、Control、Sham and OVX+CIA groups.2 Effect of Yishen Juanbi pill on joint injury in ovariectomized CIA rats2.1 Effect of Yishen Juanbi pill on arthritis index in ovariectomized CIA ratsCompared with CIA group,after 14 days of immunization,there was an upward trend in OVX+CIA group,but there was no statistical difference.After administration,the OVX+CIA+EV and the OVX+CIA+YSJB group were compared with the OVX+CIA group,and the AI decreased.The OVX+CIA+YSJB group was significantly lower than the OVX+CIA group for twenty-eighth days.The AI decreased in the OVX+CIA+EV group compared with the OVX+CIA group,but there was no statistical difference.2.2 Effect of Yishen Juanbi pill on pathological injury of knee joint in ovariectomized CIA ratsThe observation results under light microscope show that:Control and Sham groups:The joint structure was normal,the synovial tissue had no hyperemia and edema,the synovial cells were not proliferated,the articular cartilage surface was smooth and no exfoliation,the size and arrangement of the bone trabecula in the subchondral bone and the backbone were normal.OVX group:joint structure,synovial membrane and articular cartilage were not abnormal,but there were different degrees of adipocyte proliferation in the bone marrow cavity of the joint epiphysis and bone marrow.The bone trabecula became thinner and the gap was enlarged than that of Control and Sham group.CIA group:the proliferation of synovial cells in different degrees,hyperemia and edema of synovial tissue,capillary hyperplasia,infiltration of inflammatory cells,the formation of pannus in the hyperplastic synovial tissue,and the degeneration,necrosis and exfoliation of cartilage tissue.OVX+CIA group:in addition to the pathological features of CIA group,different degrees of adipocyte proliferation were seen in the bone marrow cavity of the joint epiphysis and bone marrow,which were more obvious than those in the CIA group,and the bone trabecula became thinner and the gap increased.OVX+CIA+EV group and OVX+CIA+YSJB group:pathological changes were alleviated to different degrees compared with OVX+CIA group.The results of joint pathological injury showed that the infiltration of arthritis cells and the injury of cartilage and bone in OVX+CIA group were higher than that in CIA group.Compared with OVX+CIA group,the invasion of osteoarthritis cells and the injury of cartilage and bone in OVX+CIA+YSJB group were obviously reduced;In OVX+CIA+EV group,inflammatory cell infiltration and pathological score of cartilage and bone injury decreased,but there was no statistical difference.3 Effect of Yishen Juanbi pill on bone microstructure in ovariectomized CIA ratsThere was no significant change in BV,BS and BS/BV among Control,Sham and OVX groups;compared with Control,Sham and OVX group,there was no significant difference in BV between CIA,OVX+CIA,OVX+CIA+EV and OVX+CIA+YSJB groups,but BS and BS/BV increased significantly.Compared with OVX+CIA group,the ankle joint BV of rats in OVX+CIA+YSJB group was significantly increased,and BS and BS/BV decreased obviously.The BS of ankle joint in OVX+CIA+EV group had a downward trend compared with that of OVX+CIA group,but there was no statistical difference.4 Effect of Yishen Juanbi pill on osteoclast differentiation in ovariectomized CIA rats4.1 Effect of Yishen Juanbi pill on osteoclast count in ovariectomized CIA ratsTRAP is a marker enzyme of osteoclasts,which is often used to detect osteoclast activity.After the decalcification of the knee joint,the paraffin sections of the rats were stained with TRAP to observe the quantity and distribution of osteoclasts.In this experiment,we first observed the number and distribution of TRAP positive osteoclasts in synovial tissue of rats.No TRAP staining osteoclasts were found in the synovial tissues of Control,Sham and OVX groups,but in CIA,OVX+CIA,OVX+CIA+EV and OVX+CIA+YSJB groups,there were different size and depth of TRAP stained osteoclasts.After analyzing the number of osteoclasts,it was found that the number of osteoclasts in CIA,OVX+CIA,OVX+CIA+EV and OVX+CIA+YSJB groups was significantly higher than that in Control,Sham and OVX groups.Compared with the OVX+CIA group,the number of osteoclasts in OVX+CIA+YSJB group rats decreased significantly,and the number of osteoclast in OVX+CIA+EV group decreased,but there was no statistical difference.We also found that the osteoepiphysis of the tibia and femur of the knee joint of the rat can be seen with TRAP staining positive osteoclasts,most of them on the edge of the trabecular bone.If articular cartilage is eroded by synovium,osteoclasts positive for TRAP staining in the epiphysis end are significantly increased.Compared with Control group and Sham group,the amount of osteoclast in the epiphysis and diaphysis of OVX group increased significantly,and the osteoclast in OVX+CIA+EV group and OVX+CIA+YSJB group decreased obviously than that in OVX+CIA group.The number of osteoclasts in bone epiphysis and bone marrow tissue was analyzed.Compared with Control group and Sham group,the number of osteoclasts in OVX,CIA,OVX+CIA,OVX+CIA+EV and OVX+CIA+YSJB group increased significantly.Compared with OVX group,the number of osteoclasts in OVX+CIA group,OVX+CIA+EV group and OVX+CIA+YSJB group increased significantly.The number of osteoclasts in OVX+CIA group,OVX+CIA+EV group and OVX+CIA+YSJB group was also significantly higher than that in CIA group.Compared with OVX+CIA group,the number of osteoclasts in OVX+CIA+EV group and OVX+CIA+YSJB decreased significantly.4.2 Effect of Yishen Juanbi pill on the expression of osteoclast differentiation factor NFATcl protein in synovial tissue and bone marrow tissue of ovariectomized CIA ratsImmunohistochemical method was used to detect the expression of NFATcl protein in synovial tissues and bone marrow tissues of rats.It was found that NFATcl was expressed in the nucleus.The expression is as follows:the expression of NFATcl is weak in the nonproliferative synovial tissue and the expression of NFATcl in the hyperplastic synovial tissue is enhanced,and the NFATcl staining positive multicore osteoclasts are found in the hyperplastic synovial tissue;the expression of NFATc1 in bone marrow tissue is strongly positive.The analysis of NFATcl protein in synovial tissue showed that the expression of NFATcl protein in CIA group,OVX+CIA group,OVX+CIA+EV group and OVX+CIA + YSJB group increased significantly compared with Control group,Sham group and OVX group.There was no significant difference in the expression of NFATcl protein between OVX+CIA group,OVX+CIA+EV group and OVX+CIA+ YSJB group compared with CIA group.Compared with the OVX+CIA group,the expression of NFATc1 protein in the OVX+CIAH+EV group had a downward trend,but the difference was significant.The expression of NFATcl protein in the OVX+CIA+ YSJB group rats decreased obviously,and there was no statistical difference in the expression of NFATc1 protein in the OVX+CIA+EV group compared with the OVX+ CIA+YSJB group.The analysis of NFATcl protein in bone marrow tissue showed that the expression of NFATcl protein in OVX,CIA,OVX+CIA,OVX+CIA+EV and OVX+CIA+YSJB groups increased significantly compared with the Control and Sham groups.Compared with OVX group,there was no significant difference in NFATcl protein between CIA.OVX+CIA,OVX+CIA+EV and OVX+CIA+YSJB groups.The expression of NFATcl protein in OVX+CIA group was significantly higher than that in CIA group.Compared with the OVX+CIA group,the expression of NFATcl protein in OVX+CIA+EV and OVX+CIA+YSJB groups decreased significantly,and there was no statistical difference in the expression of NFATc1 protein in OVX+CIA+EV and OVX+CIA+YSJB group.5 Effect of Yishen Juanbi pill on the expression of ephrinB2 protein in synovial tissue and bone marrow of ovariectomized CIA ratsImmunohistochemistry was used to detect the expression of ephrinB2 protein in synovial tissues and bone marrow tissues of rats.It was found that ephrinB2 was expressed in cell membrane and cytoplasm.EphrinB2 protein was expressed in a small amount in the synovial tissue,and expressed strongly in bone marrow stromal cells,osteoclasts,osteoblasts and vascular endothelial cells.The osteoclasts in the synovial and bone marrow tissues were expressed in ephrinB2.The analysis of ephrinB2 protein in synovial tissue showed that the expression of ephrinB2 protein in OVX group was significantly lower than that of Control group and Sham,and the expression of protein expression in CIA group,OVX+CIA group,OVX+CIA+EV group and OVX+CIA+YSJB group was significantly higher than that of Control group,Sham group and OVX group.There was no significant difference in the expression of ephrinB2 protein between CIA group,OVX+CIA group,OVX+CIA+EV group and OVX+CIA+YSJB group.The analysis of ephrinB2 protein in bone marrow tissue showed that the expression of ephrinB2 protein in OVX group,OVX+CIA group,OVX+CIA+EV group and OVX+CIA+YSJB group decreased significantly compared with Control,Sham and CIA groups.Compared with OVX+CIA group,the expression of ephrinB2 protein in OVX+CIA+EV and OVX+CIA+YSJB groups increased significantly.6 Effect of Yishen Juanbi pill on the expression of EphB4 protein in synovial membrane and bone marrow tissue of ovariectomized CIA ratsThe expression of EphB4 protein in the synovial and bone marrow tissues of rats was detected by immunohistochemical method.It was found that EphB4 was mainly expressed in the membrane and cytoplasm of osteoblasts,bone marrow stromal cells and synoviocytes.In synovial tissues,the expression of osteoblasts in bone marrow stromal cells,especially trabecular meshwork,was weak.The analysis of EphB4 protein in synovial tissue showed that there was no significant difference in the expression of EphB4 protein among the seven groups.The analysis of EphB4 protein in bone marrow tissue showed that:compared with Control and Sham group,the expression of EphB4 protein in OVX,CIA,OVX+CIA,OVX+CIA+EV and OVX+CIA+YSJB group decreased significantly.Compared with CIA,the expression of EphB4 protein in OVX+CIA group decreased significantly.Compared with OVX+CIA group,the expression of EphB4 protein in OVX+CIA+EV and OVX+CIA+YSJB group increased.but there was no significant difference.ConclusionYishen Juanbi pill can ameliorate the swelling of the ankle joint of the CIA rats and the destruction of the bone microstructure on the surface of the tarsal bone,and reduce the infiltration of inflammatory cells,the hyperplasia of synovial membrane and the destruction of the cartilage and bone.The mechanism is to inhibit the invasion of pannus by increasing the expression of EphrinB2 in the bone marrow,inhibiting the excessive enhancement of the differentiation and activity of osteoclast in the bone marrow,inhibiting the bone resorption;on the other hand,Yishen Juanbi pill reduced the expression of osteoclast differentiation factor NFATcl in synovial tissues,thereby inhibiting the differentiation of osteoclasts in synovial tissues.Yishen Juanbi pill can inhibit the differentiation and activity of osteoclast of articular cartilage by double regulating action of these two ways,thus reduce the articular cartilage and bone destruction. |
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