Aikeqing Granule:the Inhibitory Effect On Osteoclast Differentiation,the Protective Effect On Ovairectomy-induced Bone Loss In Rats,and The Underlying Mechanisms

ObjectivePostmenopausal osteoporosis is a metabolic bone disease in which bone mass is reduced and the microstructure of bone is degraded due to lack of estrogen.The theory of traditional Chinese medicine deems that deficiency of the kidney and spleen caused by menopause is the main cause of osteoporosis.The compound Chinese medicine formula"Aikeqing Granules",developed by the Institute of Tropical Medicine of Guangzhou University of Chinese Medicine,has the effects of invigorating the kidney and spleen,replenishing Qi and Yang,and is clinically used in the adjuvant treatment of HIV-1 infection and AIDS.In order to expand the scope of clinical application of Aikeqing,whether Aikeqing improves postmenopausal osteoporosis caused by kidney and spleen deficiency was investigated in this study.By employing both cultured cells and rats with kidney and spleen deficiency induced by ovariectomy,the effects of Aikeqing on osteoblastogensis and osteoclastogenesis,and the mechanism by which Aikeqing inhibits osteoclast formation and bone resorption were studied.Methods1.The effects of Aikeqing on bone loss in ovariectomized rats(1)Animal grouping and treatmentOvariectomized SD rats(6-8 months old)were randomly divided into five groups(N=10 in each group)and orally treated with 1 g/kg/d(ALD group),2 g/kg/d(AMD group)or 4 g/kg/d(AHD group)of Aikeqing,or 0.1 mg/kg/d of estradiol valerate(EV group),or vehicle(OVX group).Ten female rats of the same age subjecting to sham operation served as normal control(Sham group).The treatments were terminated after 3 months of continuous administration.(2)Detection,measurement and analysis①Bone tissue morphometric parameter analyses and three-dimensional structure reconstruction of the bone were performed on a Micro-CT;② Histopathological changes of the bone tissue were detected by H&E staining;③The detection as well as the number of osteoclasts in the bone tissue were performed using TRAP staining;④ Organ indexes(heart,liver,spleen,lung,kidney and uterus),liver function indexes(serum levels of ALT and AST),and renal function indexes(serum levels of CRE and BUN)were analyzed;⑤mRNA levels of bone resorption-related genes(CA2,CTSK,MMP9 and ATP6V0D2)and bone formation-related genes(BMP-2 and Sp7)in the bone tissue were analyzed by real-time PCR;⑥Expression of the bone resorption function-related proteins(Tracp,CTSK and c-fos)in bone tissue was detected by Western blotting.2.The effects of Aikeqing on the proliferation and differentiation of osteoblasts(1)The effects of different concentrations of Aikeqing on the proliferation of MC3T3-E1 cells within 48 h or 72 h were assessed by CCK-8 assay;(2)the effects of Aikeqing on the osteogenic activity of MC3T3-E1 cells were evaluated by the secreted ALP measured using an ALP kit,and the cellular ALP levels detected using an ALP staining kit.3.The effect of Aikeqing on RANKL-induced osteoclast differentiation(1)The effect of Aikeqing on the proliferation of bone marrow-derived macrophages:Monocytes extracted from the bone marrow of C57BL/6 mice were induced by M-CSF for 4 days to provide bone marrow-derived macrophages(BMMs).Then effects of Aikeqing on the proliferation of BMMs within 48 h or 72 h were detected by CCK-8 assay.(2)The effect of Aikeqing on osteoclast differentiation:BMMs were induced for osteoclast differentiation with RANKL,with or without the intervention of Aikeqing.The formed osteoclasts were detected and counted after staining the cellular TRAP using a TRAP staining kit;the F-actin rings in the osteoclasts were detected by immunofluorescence staining.(3)Measurement of the mRNA levels of marker genes in osteoclast differentiation:BMMs were induced for osteoclast differentiation with RANKL,with or without the intervention of Aikeqing.Total RNA samples were then prepared.Real time PCR analyses were employed to quantify the mRNA levels of the bone resorption-related genes(Car2,Tracp,Ctsk and Atp6v0d2),osteoclast surface receptor genes(Calcr and RANK),and the osteoclast precursor fusion genes(c-fos and Dcstamp).(4)Detection of the marker proteins in osteoclast differentiation:BMMs were induced for osteoclast differentiation with RANKL,with or without the intervention of Aikeqing.Total protein samples-were then prepared,Western blot analyses were employed to detect the expression of proteins related to bone resorption,including(Mmp9,Ctsk and Tracp).4.Inhibitory mechanism of Aikeqing in osteoclast differentiation(1)The effects of Aikeqing on the expression and nuclear translocation of key transcription factors for osteoclast differentiation:BMMs were induced for osteoclast differentiation with RANKL,with or without the intervention of Aikeqing.Cellular protein levels of transcriptional factors NFATcl and c-fos were detected by Western blotting,while nuclear translocation of the two proteins was observed by immunofluorescence staining.(2)The effects of Aikeqing on the key regulatory pathways in osteoclast differentiation:BMMs were pretreated with Aikeqing and then stimulated with RANKL for 0,5 10,20,30 and 60 min.Western blot analyses were performed to detect the expression of proteins in the NFκB pathway(p65,p-p65,IκBα and p-IκBα),the JNK pathway(JNK and p-JNK),and the p38 pathway(p38 and p-p38),at each time points.(3)The effect of Aikeqing on nuclear translocation of p65:BMMs were pretreated with Aikeqing and then stimulated with RANKL for 0,30 and 60 min.protein levels of p65 in the cytoplasm and the nuclear were analyzed by Western blotting,and the nuclear expression of p65 was observed by immunofluorescence assay.(4)The effect of Aikeqing on the interaction between RANK and TRAF6:Macrophages Raw264.7 were pretreated with Aikeqing and then stimulated with RANKL for 20 min.Co-immunoprecipitation was used to observe the TRAF6-combined RANK.Results1.The effect of Aikeqing on femur bone loss in ovariectomized rats(1)Effectively prevented ovariectomy-induced decreases in bone mineral density,bone volume fraction and trabecular number.① Micro CT scan:Compared with the Sham group,the bone mineral density(BMD),bone volume fraction(BV/TV)and trabecular number(Tb.N)of the OVX group were significantly reduced.Treatment with Aikeqing achieved dose-dependent increases in BMD,BV/TV and Tb.N.Moreover,significant increases in these indexes were observed in AHD group.② H&E staining:Compared with the Sham group,the BV/TV of the femur in the OVX group was significantly reduced.Compared with the OVX group,treatment with Aikeqing achieved dose-dependent increases in BV/TV.Moreover,significant increase in BV/TV was observed in AHD group.(2)Significantly inhibited the formation of osteoclasts in femur tissue.Osteoclasts were detected by TRAP staining.Compared with the Sham group,the osteoclast number/bone surface(N.Oc/BS)and the osteoclast surface/bone surface(Oc.S/BS)in the OVX group were increased significantly.Compared with the OVX group,the Oc.S/BS in the ALD group,the N.Oc/BS in the AMD group,and both Oc.S/BS and N.Oc/BS in the AHD group were significantly reduced.(3)Showed no adverse effects on organs.① Organ indexes:Compared with the Sham group,the organ indexes for the heart,the liver,the lung,the kidney and the uterine in the OVX group were decreased significantly;treatment with Aikeqing improved the lung index of ovarietomized rats without affecting the other organs,suggesting that Aikeqing may have beneficial effect on lung function.②Biochemical indicators of liver and kidney function:Compared with the Sham rats,OVX rats had comparable levels of AST,ALT and CRE,but significantly increased level of BUN in the sera;ompared with the OVX group,treatment with Aikeqing did not affect the serum levels of ALT,AST,and CRE but significantly decreased the serum level of BUN,suggesting that Aikeqing may have beneficial effect on renal function.(4)Significantly inhibited the expression of bone resorption-related genes.Compared with the rats in Sham group,rats in the OVX group expressed significantly upregulated mRNA levels of bone resorption-related genes(CA2,MMP9,CTSK and ATP6V0D2)but significantly downregulated mRNA levels of bone formation-related genes(BMP-2 and Sp7)in the bone tissue;Compared with rats in the OVX group,rats in the ALD group expressed downregulated mRNA levels of CA2 and MMP9,while rats in the AMD and AHD groups expressed downregulated mRNA levels of CA2,MMP9 and CTSK,without the mRNA expression of BMP-2 and Sp7 kept unchanged.It suggests that the effect of Aikeqing is to inhibit bone resorption,while not to promote bone formation.(5)Significantly inhibited the expression of bone resorption-related proteinsCompared with rats in the Sham group,rats in the OVX group expressed significantly elevated levels of bone resorption-related proteins(Tracp,Ctsk and c-fos)in the bone tissue;compared with rats in the OVX group,rats treated with Aikeqing expressed significantly decreased level of Tracp,and rats in the AHD group also expressed significantly decreased levels of Ctsk and c-fos,in the bone tissue.It further shows that Aikeqing plays a role in bone protection by inhibiting bone resorption.2.The effects of Aikeqing on the proliferation and differentiation of osteoblastsAikeqing showed inhibitory effect on the proliferation of MC3T3-E1 cells at the concentrations>50 μg/mL;while at the concentrations<50 μg/mL,Aikeqing was unable to affect either the cell proliferation or the ALP activity.These results suggest that Aikeqing has no effect on osteogenic differentiation.3.The effect of Aikeqing on the proliferation of bone marrow-derived macrophagesAikeqing,at the concentrations of 10-100 μg/mL,significantly promoted the proliferation of BMMs.4.The effects of Aikeqing on RANKL-induced osteoclast differentiation(1)Significantly inhibited the differentiation of BMMs into osteoclasts(by TRAP staining):The intervention of Aikeqing in the induction of osteoclast differentiation of BMMs reduced the number of TRAP-positive cells in a dose-dependent manner,indicating significant inhibition to osteoclastogenesis.Aikeqing,at 50 μg/mL,almost completely inhibited the formation of osteoclasts,and the inhibition was time-dependent.(2)Distinctly inhibited the formation of F-actin ring in the osteoclasts(Immunofluorescence staining):The intervention of Aikeqing in the induction of osteoclast differentiation of BMMs significantly reduced the area of F-actin ring in the osteoclasts in a dose-dependent manner and inhibited the formation of F-actin ring.(3)Remarkably inhibited the expression of bone resorption genes(Real-time PCR):The use of Aikeqing in the induction of osteoclast differentiation of BMMs downregulated the mRNA levels of bone resorption-related genes(Car2,Tracp,Ctsk,Atp6v0d2,Dcstamp and Calcr)in a dose-dependent manner;Aikeqing,at 50 μg/mL,also time-dependently downregulated the mRNA levels of bone resorption function genes(Car2,Tracp,Ctsk and Atp6v0d2),osteoclast surface receptor genes(Calcr and RANK),and osteoclast precursor fusion-related genes(c-fos and Dcstamp).(4)Significantly inhibited the expression of bone resorption proteins(Western blot):The use of Aikeqing in the induction of osteoclast differentiation of BMMs significantly reduced the expression of Mmp9,Ctsk and Tracp in a dose dependent manner;Aikeqing,at 50 μg/mL,also showed time-dependent inhibition to the expression of the three proteins.5.Mechanisms by which Aikeqing inhibits osteoclast differentiation(1)Suppressing the expression of NFATc1 and c-fos,the key transcriptional factors for osteoclast differentiation(Western blot):The intervention of Aikeqing in the induction of osteoclast differentiation of BMMs significantly reduced the expression levels of NFATc1 and c-fos,which are the key transcriptional factors,in a dose dependent manner;Aikeqing,at 50 μg/mL,also time-dependently inhibited the expression of NFATc1 and c-fos.(2)Inhibiting the nuclear translocation of NFATc1 and c-fos(Immunofluorescence staining):The intervention of Aikeqing in the induction of osteoclast differentiation of BMMs significantly reduced the expression of NFATc1 and c-fos in the nucleus,showing that Aikeqing is able to inhibit the nuclear translocation of NFATc1 and c-fos.(3)Inhibiting the activation of NFκB and MAPK which are the key pathways for osteoclast differentiation(Western blot):Induction with RANKL significantly increased the expression of p-p65,p-IκBα,p-JNK and p-p38 and decreased the expression of IκBα,JNK and p38 in BMMs,indicating the activation of NFκB pathway and MAPK pathway;The intervention of 50 μg/mL Aikeqing reduced the expression levels of p-p65,p-IκBα,p-JNK and p-p38,and increase the level of IκBα,indicating that Aikeqing antagonized RANKL-induced activation of NFκB and MAPK pathways.(4)Inhibiting the nuclear translocation of p65(Western blot and Immunofluorescence staining):RANKL stimulation caused a significant decrease in cytoplasmic p65 but a significant increase in nuclear p65 in BMMs,indicating the activation of NFκB pathway;intervention of Aikeqing(50 μg/mL)increased the cytoplasmic p65 and decreased the nuclear p65,indicating that Aikeqing antagonized RANKL-induced activation of NFκB pathway.Inhibitory effect of Aikeqing on nuclear translocation of p65 was confirmed by the results from immunofluorescence staining.(5)Inhibiting the formation of RANK-TRAF6 complex(Co-immunoprecipitation):RANKL stimulation increased the RANK-TRAF6 complexes in Raw264.7 cells;intervention of Aikeqing(50 μg/mL)significantly reduced the amount of RANK-TRAF6 complexes,indicating that Aikeqing inhibited the RANKL-induced RANK-TRAF6 interaction.Conclusion1."Aikeqing Granule",a compound Chinese medicine for invigorating the kidney and strengthening the spleen,has a protective effect on estrogen deficiency-induced bone loss in ovariectomized rats.Its function of invigorating the kidney and bone is related to the inhibition to osteoclastogenesis and bone resorption activity.2."Aikeqing Granule" significantly inhibited the formation of osteoclasts and bone resorption activity in vitro through inhibiting the RANKL-induced formation of RANK-TRAF6 complex,thereby inhibiting the activation of downstream NFκB,p38 and JNK pathways that are essential for osteoclast differentiation.3."Aikeqing Granule" has the potential to be used for the prevention and treatment of postmenopausal osteoporosis.