| Objective To evaluate the pharmacodynamic effects of the combination of Herba Epimedii and Fructus Ligustri Lucidi and active ingredients on osteoporosis(OP)rats induced by glucocorticoid(GC),and explore the mechanism of the compatibility decoction and its active ingredients in glucocorticoid-induced osteoporosis(GIOP)rats based on the regulation of TGF-beta 1/Smads signaling pathway.Methods1.Pharmacodynamic effects of the combination of Herba Epimedii and Fructus Ligustri Lucidi decoction and active ingredients on GIOP rats: 70 healthy male SD rats,randomly divided into seven groups: normal control group,GIOP model group,YN decoction high dose group(7 g/kg),YN decoction low dose group(3.5 g/kg),YN active fractions high dose group(200 mg/kg),YN active fractions low dose group(100 mg/kg)and Calcium with Vitamin D3 positive drug group(0.2773 g/kg).All rats received intramuscular injection of dexamethasone(1 mg/kg,twice a week)for 8 weeks to establish osteoporosis model except those in the control group.Rats in treatment groups were treated with intragastric administration of traditional Chinese herbs or positive drug respectively.Eight weeks later,bone mineral density(BMD)was measured by dual-energy X-ray absorptiometry.Bone biomechanical properties were detected by 3-point bending test.Serum calcium(Ca)and phosphorus(P)were detected by colorimetry.The contents of bone Ca and P were measured by Inductively coupled plasma mass spectrometry.Serum bone alkaline phosphatase(b-ALP),bone γ-carboxyglutamic acid-containing protein(BGP)and tartrate-resistant acid phosphatase(StrACP)were determined by enzyme-linked immunosorbent assay(ELISA).Morphological changes of bone tissue was detected by HE staining,2.Mechanism of decoction and active ingredients of Herba Epimedii and Fructus Ligustri Lucidi on GIOP rats: The methods of establishment of GIOP rats and administration were the same as the part one.Serum corticosterone(CORT)and glucocorticoid receptor(GR)were determined by ELISA.The content of glucocorticoid receptor and the binding force of glucocorticoid-glucocorticoid receptor in bone tissue were detected by Flow cytometry(FCM).The protein expressions of TGF-β1,Smad2/3,Smad4,and Smad7 were detected by Immunofluorescence staining(IF)and Western blot(WB).And real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect TGF-β1,Smad2,Smad3,Smad4,Smad7 mRNA expressions in bone tissue.Results1.Results of the first experiment: Compared with normal group,body weight of rats in GIOP rats were decreased significantly(P<0.01).BMD,maximum load and maximum deflection of femur were decreased significantly(P<0.01).The contents of Ca and P in bone and serum were decreased(P<0.01).Serum levels of b-ALP and BGP were inhibited significantly(P<0.01),while serum StrACP was elevated(P<0.01).The reductions in trabecular bone area(Tb.Ar),trabecular number(Tb.N)and trabecular thickness(Tb.Th)were observed in model group(P<0.01).Administration of combined decoction and combined active fractions of Herba Epimedii and Fructus Ligustri Lucidi could prevent body weight loss(P<0.01),attenuate biomechanical properties and BMD reduction(P<0.01),increase the contents of Ca and P in serum and bone(P<0.05 or P<0.01),enhance levels of serum b-ALP and BGP(P<0.05 or P<0.01),suppress serum TRACP contents(P<0.01).They could also mitigate deterioration of trabecular architecture in model rats by increasing Tb.Ar,Tb.N and Tb.Th(P<0.01),decreasing Tb.Sp(P<0.01).High dosage group of combined decoction and combined active fractions showed a better improvement than the low dosage group.2.Results of the second experiment: Compared with normal group,serum levels of CORT and GR in GIOP rats were decreased significantly(P<0.01).The contents of GR and the binding force of glucocorticoid-glucocorticoid receptor in bone tissue were increased significantly(P<0.01).Immunofluorescence staining(IF),Western blot(WB)and quantitative real-time polymerase chain reaction(qRT-PCR)analysis showed that the proteins and mRNA expressions of TGF-β1,Smad2,Smad3 and Smad4 were down-regulated respectively(P<0.05 or P<0.01),but the level of Smad7 was up-regulated(P<0.01).After administration of Herba Epimedii and Fructus Ligustri Lucidi decoction and active fractions,the serum levels of CORT and GR showed a clear upward trend(P<0.05 or P<0.01).The contents of GR and the binding force of glucocorticoid-glucocorticoid receptor in bone tissue showed a clear downward trend(P<0.05 or P<0.01).This combined herbs could also increase the expressions of TGF-β1,Smad2,Smad3 and Smad4(P<0.05 or P<0.01),while decrease the expression of Smad7(P<0.01).Conclusions1.Combined decoction and combined active fractions of Herba Epimedii and Fructus Ligustri Lucidi play a role in anti-osteoporosis through the following ways: reversing the state of low conversion type of bone metabolism induced by GC effectively,adjusting the coupling balance between bone formation and resorption,increasing BMD and bone biomechanical properties,reversing pathological changes in bone tissue.2.By reducing the expression of GR and inhibiting the binding force between glucocorticoid and glucocorticoid receptor in bone tissue,Herba Epimedii and Fructus Ligustri Lucidi could reduce the harm of GC on bone.At the same time,these combined herbs could obviously improve the inhibitory effect of GC on serum CORT and GR and increase the sensitivity of hormones,so as to have a good protective effect on the endogenous GC pathway,reduce the dosage of hormone,and prevent the occurrence of GIOP.3.The mechanism of the compatibility decoction and its active ingredients of anti-osteoporosis may be related to the regulation of TGF-β1/Smads signal transduction pathways.4.Considering the effect of each index,the effect of combined decoction and combined active fractions of Herba Epimedii and Fructus Ligustri Lucidi on prevention and treatment of GIOP is superior to Calcium with Vitamin D3 group.And among them,the improvement of YN active fractions high dose group,YN decoction high dose group is better.The compatibility active fractions have the good effects in prevention and treatment of GIOP as compatibility decoction. |
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