| ObjectiveOur previous animal experimental study shows that Yishen Juanbi Pill can improve the swelling degree of ankle joint and the destruction of bone microstructure on the surface of tarsal bone in ovariectomized rats with Collagen Induced Arthritis(CIA),and reduce the infiltration of inflammatory cells,synovial hyperplasia and the destruction of cartilage and bone in local joints.The mechanism is closely related to upregulation of ephrinB2 protein,inhibition of differentiation and excessive enhancement of activity of Osteoblast(OC).On this basis,this study reveals the mechanism of Yishen Juanbi Pill regulating the differentiation and function of osteoclasts from the perspective of ephrinB2 signal pathway by preparing the serum containing Yishen Juanbi Pill,a CIA model of kidney deficiency(ovariectomy),and inducing osteoclasts in vitro by using mouse spleen mononuclear cells and bone marrow mononuclear macrophages,in order to provide further experimental basis for the theory of "kidney governing bone" and "treating rheumatoid arthritis from kidney".Methods1 preparation of drug-containing serumThirty-five SPF female Sprague-Dawley(SD)rats were randomly divided into three groups according to their body weight after adaptive feeding:Control group(5),CIA group(7)and ovariectomized group(23).Twelve weeks later,five ovariectomized rats were randomly selected as OVX group,and other ovariectomized rats and CIA group rats were immunized with type II collagen to make CIA model.After 14 days of immunization with type II collagen,the rats without disease were eliminated.Ovariectomized CIA rats were randomly divided into three groups according to joint swelling degree:ovariectomized CIA(OVX+CIA)group(6),ovariectomized CIA estradiol valerate(OVX+CIA+EV)group(6)and ovariectomized CIA Yishen Juanbi Pill(OVX+CIA+YSJB)group(6).OVX+CIA+YSJB group rats were given 0.3g/mL Yishen juanbi pill aqueous solution,and OVX+CIA+EV group rats were given 0.0184mg/ml estradiol valerate aqueous solution,the volume of which was 1ml/100g,twice a day for five consecutive days.Control group,OVX group,CIA group and OVX CIA group were given the same volume of purified water according to the above procedure.After the last administration of water for 1 hour,anesthetize,collect blood through abdominal aorta under aseptic condition,centrifuge(2500r/min,4℃,25min),separate serum,filter and sterilize,and store at-80℃ for later use.2 the differentiation of osteoclasts induced by mouse spleen monocytes and the detection of related indexesAseptically extracting mouse spleen mononuclear cells from C57BL/6 mice for 7-8 weeks,complete medium(10%FBS)culture,is planted on a 96-well culture plate,a 96-well culture plate preset with bone slices(5×105 cells/well)and a 24-well culture plate(3×106 cells/well).Discard superlatant after 2h and add differentiation medium(25ng/ml M-CSF,50ng/ml RANKL)to each well for continuous culture.After 96 hours,the differentiation medium containing 5%drug-containing serum was added to continue to culture.After 48 hours of administration:96-well plate cells were collected for TRAP staining,and the number of TRAP osteoclasts was counted.The 24-well plate cells were collected,and the mRNA expression of transcription regulatory factors(c-Fos,c-Jun,Nfatcl,Rank,Efnb2)mRNA OC differentiation was detected by Real-time PCR.The 96-well culture plate pre-set with bone slices was cultured in compositional medium until the 11th day.The bone slices were collected and stained with toluidine blue.The morphology of bone lacunae was observed and the area of bone lacunae was analyzed by Leica Qwin image analysis system.3 Detection of osteoclasts induced by mouse bone marrow macrophages and related indexesBone marrow cells of femur and tibia of C57BL/6 mice aged 7-8 weeks were aseptically extracted.After 24 hours,the unattached suspension cell fluid was collected,cultured with differentiation medium(25ng/ml M-CSF),and planted on 96-well culture plate,96-well culture plate with bone slices(5×104 cells/well)and a 24-well culture plate(3×105 cells/well).After 72 hours of attachment,it was changed to differentiation medium(25ng/ml M-CSF,50ng/ml RANKL)containing 5%drug-containing serum.After 48 hours of administration:96-well plate cells were collected for TRAP staining,and the number of TRAP osteoclasts was counted.The 24-well plate cells were collected,and the mRNA expression of transcription regulatory factors(c-Fos,c-Jun,Nfatc1,Rank,Efnb2)mRNA OC differentiation was detected by Real-time PCR.The 96-well culture plate pre-set with bone slices was cultured in compositional medium until the 11th day.The bone slices were collected and stained with toluidine blue.The morphology of bone lacunae was observed and the area of bone lacunae was analyzed by Leica Qwin image analysis system.4 Detection of ephrinB2 gene knock-out by osteoclasts and related indexesAccording to the above method,the two kinds of cells were extracted and cultured,and transfected after 96 hours.After adding Ephrinb2 siRNA for 6 hours,they were changed into differentiation medium containing 5%drug serum and cultured for 48 hours.The 96-well plate cells were collected for TRAP staining,observed and photographed,and the number of TRAP osteoclasts was counted.The mrna expression of transcription regulatory factors(c-Fos,c-Jun,Nfatcl,Rank)mRNA OC differentiation was detected by Real-time PCR in 24-well plate Pre-prepared bone slices were cultured in the changing culture medium until the 11th day.Bone slices were collected and stained with toluidine blue.The shape of bone lacunae was observed by taking pictures,and the area of bone lacunae was analyzed by Leica Qwin image analysis system.Results1.Effect of Yishen Juanbi Pill on OC differentiation and function(spleen origin)1.1 Effect of Yishen Juanbi Pill on OC differentiation numberCompared with the blank control serum group,the number of osteoclasts in ovariectomized CIA serum group increased significantly(P<0.05);There is an increasing trend in ovariectomized serum group and CIA serum group,but there is no statistical difference(P>0.05).Compared with ovariectomized CIA serum group,the number of osteoclasts in ovariectomized CIA serum group and ovariectomized CIA Yishen Juanbi Pill serum group decreased significantly(P<0.05).1.2 Effect of Yishen Juanbi Pill on bone erosion caused by OC differentiationCompared with the blank control serum group,the area of bone resorption lacunae caused by osteoclasts in ovariectomized serum group and ovariectomized CIA serum group increased significantly(P<0.05,P<0.01);CIA serum group showed an increasing trend,but there was no statistical difference.Compared with the ovariectomized CIA serum group,the ovariectomized CIA serum group with estradiol valerate and the ovariectomized CIA serum group with Yishen Juanbi pill significantly reduced the area of bone resorption lacunae caused by osteoclasts(P<0.05,P<0.01).1.3 Effect of Yishen Juanbi Pill on mRNA expression of transcriptional regulatory factors related to OC differentiationCompared with the blank control serum group,the expression of c-Fos mRNA in ovariectomized CIA serum group was significantly higher(P<0.01).There is an increasing trend in ovariectomized serum group and CIA serum group,but there is no statistical difference(P>0.05).Compared with the ovariectomized CIA serum group,the serum group of ovariectomized CIA estradiol valerate and ovariectomized CIA Yishen Juanbi Pill showed a decreasing trend of c-Fos mRNA expression,but there was no statistical difference(P>0.05).Compared witih the blank control serum group,the expression of c-Jun mRNA in ovariectomized CIA serum group increased significantly(P<0.01);There is an increasing trend in ovariectomized serum group and CIA serum group,but there is no statistical difference(P>0.05).The expression of c-Jun mRNA in ovariectomized CIA serum group was significantly higher than that in ovariectomized CIA serum group and CIA serum group(P<0.01).Compared with the ovariectomized CIA serum group,the expression of c-Jun mRNA in the ovariectomized CIA Yishen Juanbi Pill serum group decreased significantly(P<0.05),while the ovariectomized CIA estradiol valerate serum group showed a downward trend,but there was no statistical difference(P>0.01).Compared with the blank control serum group,Nfatc1 mRNA expression in ovariectomized serum group,CIA serum group and ovariectomized CIA serum group increased significantly(P<0.05,P<0.01);The expression of Nfatc1 mRNA in ovariectomized CIA serum group was significantly higher than that in ovariectomized and CIA serum groups(P<0.05).Compared with the ovariectomized CIA serum group,the expression of Nfatc1 mRNA in ovariectomized CIA serum group and ovariectomized CIA Yishen Juanbi pill serum group decreased significantly(P<0.05,P<0.01).Compared with blank control serum group,Rank mRNA expression in ovariectomized CIA serum group increased significantly(P<0.01);Rank mRNA expression in ovariectomized CIA serum group was significantly higher than that in ovariectomized CIA serum group and CIA serum group(P<0.01).Compared with the ovariectomized CIA serum group,the expression of Rank mRNA in ovariectomized CIA serum group and ovariectomized CIA Yishen Juanbi pill serum group decreased significantly(P<0.05,P<0.01).1.4 The effect of Yishen juanbi pill on the expression of of Efnb2(encoding ephrinb2)mRNAThe expression of Efnb2 mRNA in osteoclasts in ovariectomized CIA serum group was significantly lower than that in blank control serum group(P<0.01)Ovariectomized serum group and CIA serum group showed a downward trend,but there was no statistical difference.Compared with ovariectomized CIA serum group,the expression of Efnb2 mRNA in osteoclasts in ovariectomized CIA estradiol valerate serum group and ovariectomized CIA Yishen Juanbi pill serum group was significantly higher(P<0.05).2 Effect of Yishen Juanbi Pill on OC differentiation and function(Bone marrow origin)2.1 Effect of Yishen Juanbi Pill on OC differentiation numberCompared with the blank control serum group,the number of osteoclasts in ovariectomized serum group and ovariectomized CIA serum group increased significantly(P<0.05,P<0.01);Compared with ovariectomized CIA serum group,the number of osteoclasts in ovariectomized CIA serum group and ovariectomized CIA Yishen Juanbi Pill serum group decreased significantly(P<0.05).2.2 Effect of Yishen Juanbi Pill on bone erosion caused by OC differentiationCompared with the blank control serum group,the area of bone resorption lacunae caused by osteoclasts in ovariectomized serum group and ovariectomized CIA serum group increased significantly(P<0.05,P<0.01);CIA serum group showed an increasing trend,but there was no statistical difference.Compared with the ovariectomized CIA serum group,the ovariectomized CIA serum group with estradiol valerate and the ovariectomized CIA serum group with Yishen Juanbi pill significantly reduced the area of bone resorption lacunae caused by osteoclasts(P<0.05).2.3 Effect of Yishen Juanbi Pill on mRNA expression of transcriptional regulatory factors related to OC differentiationCompared with the blank control serum group,the expression of c-Fos mRNA in ovariectomized CIA serum group increased significantly(P<0.01);There is an increasing trend in ovariectomized serum group and CIA serum group,but there is no statistical difference.Compared with the ovariectomized CIA serum group,the serum group of ovariectomized CIA estradiol valerate and ovariectomized CIA Yishen Juanbi Pill showed a decreasing trend of c-Fos mRNA expression,but there was no statistical difference.Compared with the blank control serum group,the expression of c-Jun mRNA in ovariectomized CIA serum group increased significantly(P<0.01);There is an increasing trend in ovariectomized serum group and CIA serum group,but there is no statistical difference.The expression of c-Jun mRNA in ovariectomized CIA serum group was significantly higher than that in ovariectomized CIA serum group and CIA serum group(P<0.01).Compared with ovariectomized CIA serum group,the expression of c-Jun mRNA in ovariectomized CIA serum group and ovariectomized CIA Yishen Juanbi pill serum group decreased significantly(P<0.05).Compared with the blank control serum group,Nfatc1 mRNA expression in ovariectomized serum group,CIA serum group and ovariectomized CIA serum group increased significantly(P<0.05,P<0.01);The expression of Nfatcl mRNA in ovariectomized CIA serum group was significantly higher than that in ovariectomized CIA serum group and CIA serum group(P<0.01).Compared with the ovariectomized CIA serum group,the expression of Nfatcl mRNA in ovariectomized CIA serum group and ovariectomized CIA Yishen Juanbi Pill serum group decreased significantly(P<0.01).Compared with the blank control serum group,the expression of Rank mRNA in ovariectomized serum group and ovariectomized CIA serum group increased significantly(P<0.05,P<0.01);The expression of Rank mRNA in ovariectomized CIA serum group was significantly higher than that in ovariectomized CIA serum group and CIA serum group(P<0.05).Compared with ovariectomized CIA serum group,the expression of Rank mRNA in ovariectomized CIA serum group and ovariectomized CIA Yishen Juanbi pill serum group decreased significantly(P<0.05).2.4 The effect of Yishen juanbi pill on the expression of of Efnb2(encoding ephrinb2)mRNAThe expression of Efnb2 mRNA in osteoclasts in ovariectomized CIA serum group was significantly lower than that in blank control serum group(P<0.01)Ovariectomized serum group and CIA serum group showed a downward trend,but there was no statistical difference.Compared with ovariectomized CIA serum group,the expression of Efnb2 mRNA in osteoclasts in ovariectomized CIA estradiol valerate serum group and ovariectomized CIA Yishen Juanbi pill serum group was significantly higher(P<0.05).3.The effect of ephrinB2 on regulating OC differentiation and function(spleen origin)by Yishen Juanbi Pill3.1 The effect of ephrinB2 on regulating OC number of Yishen Juanbi PillsCompared with NC siRNA ovariectomized CIA serum group,the number of osteoclasts in ephrinB2 siRNA ovariectomized CIA serum group increased significantly after transfection of ephrinB2 siRNA.Compared with NC siRNA ovariectomized CIA serum group(P<0.01),the number of OC in NC siRNA ovariectomized CIA Yishen juanbi pill serum group decreased significantly(P<0.05).Compared with ephrinB2 siRNA ovariectomized CIA Yishen Juanbi Pill serum group,the number of osteoclasts in ephrinB2 siRNA ovariectomized CIA serum group decreased,but there was no statistical difference(P>0.05).3.2 Effect of ephrinB2 on regulating OC-induced bone erosion by Yishen Juanbi PillCompared with NC siRNA ovariectomized CIA serum group,ephrinB2 siRNA ovariectomized CIA serum group significantly increased the area of bone resorption lacunae caused by osteoclasts(P<0.05).Compared with NC siRNA ovariectomized CIA serum group,the area of bone resorption lacunae caused by osteoclasts in NC siRNA ovariectomized CIA Yishen Juanbi Pill serum group was significantly reduced(P<0.01).After transfection of ephrinB2 siRNA,compared with ephrinB2 siRNA ovariectomized CIA Yishen Juanbi Pill serum group,the area of bone resorption lacunae caused by osteoclasts decreased,but there was no statistical difference(P>0.05).3.3 Effect of ephrinB2 on mRNA expression of transcription regulatory factors related to osteoclast differentiation regulated by Yishenjuanbi PillThe expressions of c-Fos,c-Jun,Nfatcl and Rank mRNA in ephrinB2 siRNA ovariectomized CIA serum group were significantly higher than those in NC siRNA ovariectomized CIA serum group(P<0.05,P<0.01).Compared with NC siRNA ovariectomized CIA serum group,the expressions of c-Jun,Nfatcl and Rank mRNA in NC siRNA ovariectomized CIA Yishen juanbi pill serum group decreased obviously(P<0.05),and c-Fos showed a downward trend,but there was no statistical difference(P>0.05).The expression of c-Fos,c-Jun,Nfatcl and Rank mRNA in ephrinB2 siRNA ovariectomized CIA Yishen juanbi pill serum group decreased compared with ephrinB2 siRNA ovariectomized CIA serum group,but the difference was not obvious(P>0.05).4.Effect of ephrinB2 on regulating OC differentiation and function(bone marrow origin)by Yishen Juanbi Pill4.1 The effect of ephrinB2 on regulating OC number of Yishen Juanbi PillsCompared with NC siRNA ovariectomized CIA serum group,the number of OC in NC siRNA ovariectomized CIA Yishen juanbi pill serum group decreased significantly(P<0.05).Compared with NC siRNA ovariectomized CIA serum group,the number of osteoclasts in ephrinB2 siRNA ovariectomized CIA serum group increased significantly(P<0.01).After transfection of ephrinB2 siRNA,the number of osteoclasts in ephrinB2 siRNA ovariectomized CIA Yishen Juanbi Pill serum group decreased compared with ephrinB2 siRNA ovariectomized CIA serum group,but there was no statistical difference(P>0.05).4.2 Effect of ephrinB2 on regulating OC-induced bone erosion by Yishen Juanbi PillCompared with NC siRNA ovariectomized CIA serum group,ephrinB2 siRNA ovariectomized CIA serum group significantly increased the area of bone resorption lacunae caused by osteoclasts(P<0.05).Compared with NC siRNA ovariectomized CIA serum group,the area of bone resorption lacunae caused by osteoclasts in NC siRNA ovariectomized CIA Yishen Juanbi Pill serum group was significantly reduced(P<0.05).After transfection of ephrinB2 siRNA,compared with ephrinB2 siRNA ovariectomized CIA Yishen Juanbi Pill serum group,the area of bone resorption lacunae caused by osteoclasts decreased,but there was no statistical difference(P>0.05).4.3 Effect of ephrinB2 on mRNA expression of transcription regulatory factors related to osteoclast differentiation regulated by Yishenjuanbi PillThe expressions of c-Fos,c-Jun,Nfatcl and Rank mRNA in ephrinB2 siRNA ovariectomized CIA serum group were significantly higher than those in NC siRNA ovariectomized CIA serum group.Compared with NC siRNA ovariectomized CIA serum group(P<0.05,P<0.01),the expressions of c-Jun,Nfatcl and Rank mRNA in NC siRNA ovariectomized CIA Yishen juanbi pill serum group decreased obviously(P<0.05),and c-Fos showed a downward trend,but there was no statistical difference(P>0.05).The expression of c-Fos,c-Jun,Nfatcl and Rank mRNA in ephrinB2 siRNA ovariectomized CIA Yishen juanbi pill serum group decreased compared with ephrinB2 siRNA ovariectomized CIA serum group,but the difference was not obvious(P>0.05).Conclusion(1)yishenjuanbi pill can up-regulate the expression of ephrinB2,down-regulate the expression of c-Fos,c-Jun,Nfatcl and Rank,and inhibit the differentiation and function of osteoclast precursors from mouse bone marrow and spleen.(2)Knocking out ephrinB2 gene can weaken the regulation of Yishen Juanbi Pill on osteoclast differentiation/function and the expression of osteoclast transcription factors c-Fos,c-Jun,Nfatcl and Rank,which indicates that ephrinB2 signal pathway is one of the key links of Yishen Juanbi Pill in inhibiting osteoclast differentiation and function. |
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