Effect And Mechanism Of Armillaria Mellea On Osteoporosis In Ovariectomized Rats And Osteoblasts

Objective: Osteoporosis is a systemic progressive disease of the skeletal system.Studies have suggested that the differentiation,proliferation and mineralization of osteoblasts are closely related to the rate of bone formation,and they are important target cells for the treatment of osteoporosis.Armillaria mellea(AM)has many biological activities,but its role in bone metabolic diseases has not been reported.The objective of this study was to investigate the prevention and treatment effect of armillaria mellea on osteoporosis and its related mechanism.Methods:(1)In vivo experiment: The SPF healthy SD rats were randomly divided into SHAM group,OVX group,Pentanoic acid estradiol(E2)group,Armillaria mellea high-dose(AMH)group,Armillaria mellea medium-dose(AMM)group,and armillaria mellea low-dose(AML)group,with 8 rats in each group.After weighing and anesthesia,only a small amount of adipose tissue around the ovary was removed in the SHAM group,and ovariectomy was performed in the other groups to establish a rat osteoporosis model.Each group was given the corresponding drug by intragastric administration,once a day,for 3 months.After the experiment,the changes of uterus morphology and lipid metabolism in ovariectomized rats were observed by measuring uterus weight,observing its morphology and calculating uterine index.Micro-CT examination of the right femur was performed to observe the changes of bone density and trabecular microstructure in each group.The maximum load,stress,strain and other biomechanical properties of femur in each rats were measured by three-point bending test.the levels of bone metabolism,inflammation and other related indexes in serum of rats were measured by elisa kit after blood collection through abdominal aorta.These indicators include bone-specific alkaline phosphatase,tartrate resistant acid phosphatase 5b,procollagen typeⅠ N-terminal propeptide,estrogen and Anti-tumour Necrosis Factor Alpha.(2)In vitro experiment:Primary rat osteoblasts were extracted by enzyme digestion method,and their morphology was observed under microscope,and identified by ALP and alizarin red staining.Distilled water and armillaria mellea aqueous solution were introduced into the rats’ stomach to prepare drug-containing serum.The effects of different concentrations of armillaria mellea-containing serum on OB proliferation were detected by MTT and Ed U methods.The activity of Alkaline phosphatase(ALP)in OB was determined by colorimetric method.Alizarin red staining was used to determine the influence of different concentrations of medicated serum on OB mineralization nodules.Western Bloting was used to detect the changes of AMPKα and ERα protein expression levels in OB of each group,so as to explore whether armillaria mellea can improve osteoporosis by regulating estrogen metabolism and osteogenic differentiation.Results:(1)In vivo experiment: OVX group rats had increased body mass,uterine atrophy,decreased uterine index and enhanced lipid metabolism.Biochemical test results showed that the levels of PINP,BALP and ER in OVX group were decreased,while the levels of TRACP-5b and TNFα were increased in the serum of OVX rats.Micro-CT scanning results showed a decrease in bone density and deterioration of bone microstructure in OVX rats.The results of three-point bending test showed that the biomechanical properties of femur in OVX rats were decreased.Compared with OVX group,uterine index,bone metabolic indexes in serum,bone mineral density,bone microstructure parameters and bone biomechanical properties of rats in all administration groups,especially those in E2 and AMH groups,were significantly improved,which preliminarily indicated that armillaria mellea could prevent and treat osteoporosis of ovariectomized rats by improving bone metabolism,promoting estrogen secretion and inhibiting inflammatory response.(2)In vitro experiments: The proliferation of OB was determined by MTT and Ed U methods,suggesting that the serum with high and medium concentrations of armillaria mellea could significantly promote the proliferation of OB.Alkaline phosphatase activity assay suggested that high concentration of armillaria mellea medicated serum could significantly promote the alkaline phosphatase activity of OB.Alizarin red staining showed that high and medium concentration of serum containing armillaria mellea could increase the number and area of OB mineralization nodules.Western Bloting results showed that high concentration of armillaria mellea drug-containing serum significantly increased the expression levels of AMPKα and ERα proteins in OB,suggesting that the ERα-AMPK pathway was involved in the process of armillaria mellea promoting differentiation,proliferation and mineralization of OB.Conclusions: Armillaria mellea can promote OB differentiation to prevent and treat osteoporosis in ovariectomy rats,and its mechanism is related to the regulation of armillaria mellea on bone metabolism,estrogen secretion,oxidative stress,etc.In addition,armillaria mellea may play an anti-osteoporosis role through the ER-α-AMPK signaling pathway.This study provides in vivo and in vitro experimental basis for armillaria mellea prevention and treatment of osteoporosis,and provides a new direction for clinical research and development of anti-osteoporosis preparations.