Aqueous Extract Of Salvia Miltiorrhiza Bunge-Radix Puerariae Herb Pair Attenuates Osteoporosis In Ovariectomized Rats Through Suppressing Osteoclast Differentiation

Objective:Osteoporosis is a systemic progressive skeletal disease,and the main cause of osteoporosis is excessive bone resorption due to osteoclast differentiation,making osteoclast（OC）an important target cell for the treatment of osteoporosis.Salvia miltiorrhiza Bunge-Radix Puerariae（DG）has several biological activities and its role in skeletal disorders has not been reported.The aim of this paper is to investigate the role of DG in osteoporosis and related mechanisms.Methods:First of all,network pharmacology was used to predict whether DG had the potential to improve osteoporosis.In the subsequent in vivo experiments,40 female SD rats were randomly assigned into sham group,vehicle group,DG low dosage group and DG high dosage group,with 10 rats in each group.Each rat was weighted,anesthetized with10%chloral hydrate via intraperitoneal injection.The sham group animals received laparotomy of both sides of abdominal skin and muscle only,while other animals received ovariectomy（OVX）on both sides.Drugs were administered via orally treatment based on body weight one day after the surgery.Animals in sham group and vehicle group received saline,while DG low dose group received 50 mg/kg and DG high dose group received 200mg/kg of DG.After 8 weeks,Bone tissues were collected for H&E staining to assess the morphology,TRAP staining was performed to observe the differentiation of osteoclasts,and immunohistochemistry was utilized to measure the expression levels of cathepsin K and NFATC1.Meanwhile,the tibia of rats was collected and subjected to Micro-CT examination.Bone density and bone trabecular structure were analyzed in each group.Serum of rats were collected,and the serum levels of RANKL,OPG,ALP,BUN and creatinine were measured with biochemical kit.Additionally,RANKL was used to induce RAW264.7 cells to differentiate into osteoclasts in our in vitro experiments.TRAP staining was used to confirm the differentiation of osteoclasts.Western Blot was used to assess the expression levels of osteoclast differentiation markers c-Fos and NFATC1.In order to further explore the mechanism,parameters related to autophagy and oxidative stress were investigated.Autophagy levels in cells stimulated with RANKL were detected via Western Blot and MDC staining,and then the expression levels of autophagy related proteins p62,Beclin-1 and LC3B-Ⅱ/Ⅰfollowing DG treatment were further measured with Western Blot.To determine the effect of DG on oxidative stress induced by RANKL stimulation,the intracellular levels of ROS and Ca²⁺were detected with flow cytometry.Furthermore,autophagy inhibitor,ROS scavenger,Ca²⁺chelator and NF-κB inhibitor were exposed to the cells,and the relationships among DG and autophagy/oxidative stress/NF-κB were detected with Western Blot.Results:First of all,the results of the network pharmacological analysis showed that DG is closely associated with inflammatory diseases as well as metabolic diseases,and NF-κB is its important target,presumably DG has potential preventive and curative effects on osteoporosis.Then H&E staining,Masson staining and Micro-CT results indicated bone destruction,microarchitectural deterioration and decreased bone density of femur bone in the vehicle group,while DG treatment significantly alleviate these changes,confirming DG’s potential in osteoporosis treatment as suggested by network Pharmacology.Furthermore,DG treatment restored the serum levels of osteoclast-specific markers（RANKL,ALP and OPG）as well as decreased NFATc1-positive osteoclasts in vehicle group.These data suggested that possible mechanisms for DG-mediated osteoporosis alleviation was to suppress osteoclast differentiation.Chronic renal damage is one of the common serious adverse effects following most clinically-available osteoporosis drug treatments.In the current study,renal function was measured in the animals.Intriguingly,DG treatment improved renal function comparing to those of vehicle group,suggesting potential advantages in clinical applications.Furthermore,in vitro experiments revealed that RANKL-induced osteoclast differentiation was extensively suppressed by DG pretreatment.The presence of autophagosome and expression of autophage markers LC3B-II/I confirmed autophagy in the cells following RANKL stimulation,which can be reversed by DG pretreatment.Besides,DG effectively reduced RANKL-induced ROS production.ROS scavenger,Ca²⁺chelator and NF-κB inhibitor all significantly suppressed osteoclast differentiation.Conclusion:In summary,DG treatment may ameliorate osteoporosis by regulating osteoclast differentiation mediated by autophagy and oxidative stress.This study provided a mechanistic basis for utilizing DG for osteoporosis treatment,and offered a safe dose for DG in bone diseases prevention and improvement.