Liensinine Reduces Bone Loss In Ovariectomized Mice Through Promoting Osteoclast Apoptosis

Background:Osteoporosis?Osteoporosis,OP?is a systemic inflammatory bone disease,also belongs to a metabolic disease,which is characterized by the decrease of bone mass and bone mineral density and the deterioration of bone tissue microstructure,which leads to the complications of increased bone fragility and increased susceptibility to fracture^[1].In primary osteoporosis,postmenopausal osteoporosis?Postmenopausal osteoporosis,PMOP?is the most common type.The incidence of postmenopausal osteoporosis remains high in elderly women,which generally occurs within 5 to 10 years after menopause.Because it is easy to lead to cone and hip fractures,it not only reduces the individual quality of life,but also brings huge economic and human burden to the society^[2-3].The high level of bone remodeling and the disorder between bone resorption and osteogenesis are considered to be the main pathogenesis of PMOP^[4].Sex steroids play a key role in maintaining the homeostasis of the skeletal system,and the bone fracture rate of vertebral body and hip is greatly reduced after estrogen replacement therapy in postmenopausal women^[5].The inhibitory effect of estrogen on bone loss is mainly reflected in the following two aspects:first,estrogen can directly reduce the number of osteoclasts in bone,affectting the shape and size of osteoclasts and decrease their activity.Secondly,estrogen can increase the expression of apoptosis-related factor ligand?Fas L?in osteoblasts,and induce the apoptosis of osteoclasts by enhancing the interaction between osteoblasts and osteoclasts^[7].After menopause,the decrease of estrogen in the circulation leads to the increase of osteoclast production,the decrease and postponement of mature osteoclast apoptosis,the decrease and postponement of mature osteoclast apoptosis,and the prolongation of osteoclast life cycle.The bone resorption function of osteoclasts was prolonged,at the same time,the formation and recruitment of osteoblasts were hindered,the role of bone formation was limited,and the balance between bone resorption and bone production was broken.Resulting in a large number of postmenopausal bone loss,bone mineral density decreased to cause osteoporosis^[8].Increasing evidences showed that the prolongation of osteoclast life cycle plays an important role in the imbalance of bone remodeling^[9].Therefore,we believe that reducing osteoclast-mediated bone resorption by promoting apoptosis and shortening the lifespan of mature osteoclasts is another promising strategy for the prevention and treatment of PMOP.Clinically,for the treatment of osteoporotic fracture,the priority is to treat osteoporosis first and then operation,and continuing the osteoporosis treatment.For the treatment of osteoporosis,diet is as important as medicine.After screening and pharmacological analysis of patients'dietary materials,we found that Nelumbinis Plumula is loved by patients and their families in terms of both tea and food.With the ingredients include alkaloids,flavonoids and water-soluble polysaccharides,Nelumbinis Plumula has the effect of soothe the nerves,anti-oxidation,anti-inflammation and hemostasis.Liensinine,the main active ingredient in Nelumbinis Plumula,is a kind of dibenzyl tetrahydroisoquinoline alkaloid.^[10].Liensinine is one of the most active small monomers of traditional Chinese medicine extracted from Nelumbinis Plumula.Studies have shown that liensinine has many pharmacological effects,such as promoting tumor cell apoptosis,anti-inflammation,antioxidation and so on^[11].It can be used in the treatment of digestive tract tumors and has an inhibitory effect on the invasion of thymic tumors,especially on bone invasion of thymic tumors^[12].In view of the pharmacological effects of liensinine,such as anti-inflammation and promoting tumor cell apoptosis,we believe that liensinine is a small molecular monomer of traditional Chinese medicine which can alleviate bone loss,and it is expected to be a safe and effective choice for the treatment of osteoporosis.The purpose of this study is to make an observation that whether the liensinine could have any effects on bone lose in mice model of POMP and its underlying mechanism.Objective:1.To analyze the the effect of liensinine on bone tissue of ovariectomized mice by vivo experiment.2.To analyze the effects of liensinine on the differentiation,function and survival of osteoclasts,under the in vitro experiment circumstances.3.To analyze the molecular mechanism of the effect of liensinine on bone and osteoclasts in vitroMethods:Part 1:Effects of liensinine on bone loss,serum osteoclast,osteogenesis and inflammation in in ovariectomized mice.Adult mice?Type:C57BL/6,Sex:female,Age:6-8 weeks?were divided into three groups,randomly.?1?No ovariectomy group?Sham group?;?2?ovariectomized osteoporosis model group?blank control group OVX?;?3?POMP model+liensinine experimental treatment group?experimental group OVX+Lie?,6 rats in each group?n=6?.Among them,the sham operation group only performed skin incision and suture on the back of the experimental mice without ovariectomy,while the other two groups underwent ovariectomized?OVX?.From 48 hours after operation,the osteoporosis model blank control group?OVX group?received intraperitoneal injection of 0.2ml 0.9%normal saline every day,and the experimental group received intraperitoneal injection of 0.2ml liensinine?Liensinine??0.9%saline every day?.The injection dose of liensinine was 30mg/kg,and the experimental time was 6 weeks.Six weeks later,the bilateral femurs of mice were taken,and one side of the femur was decalcified,and then embedded.After,paraffin sections were made and stained with Hype and Trap.The other side of the femur was fixed with paraformaldehyde for 3 days and washed with PBS for Micro CT examination.After taking blood from the ophthalmic vein of mice,the serum samples were obtained by static centrifugation and detected by ELISA test to detect the expression levels of markers related to osteoclast,osteogenesis and aseptic inflammation in vivo.Part 2:Effects of liensinine on differentiation,function and survival of osteoclasts,under the in vitro experiment circumstances.The cytotoxicity experiment of liensinineo on mononuclear macrophage cell line RAW264.7 and primary mouse bone marrow mononuclear cells?Bone marrow mononuclear cells,BMMCs?were carried out by CCK8 test.RANKL+M-CSF were used as induction conditions to induce bone marrow mononuclear cells?BMMCs?to differentiate into osteoclasts under the in vitro experimental circumstances.As to RAW264.7,only RANKL was enough in terms of the induction into osteoclasts in vitro.In this part,we designed five groups to carry out the experiment.?1?blank control group,that is,uninduced differentiation group;?2?induced differentiation group;?3?induced differentiation+low concentration liensinine group?1?M?;?4?+medium concentration liensinine group?2?M?;?5?induced differentiation+high concentration liensinine group?4?M?.The number and morphology of differentiated osteoclasts were detected by TRAP staining,tartrate-resistant acid phosphatase staining,and the effect of liensinine on actin ring formation of osteoclasts at different concentrations was determined by osteoclast actin ring staining?F-actinring?.After the same number of differentiated mature osteoclasts were injected into the bone plate,which was made of hydroxyapatite.Liensinine of different concentrations,?1?M?2?M?4?M?,were introduced to analyze the their effects on the number and area of bone lacunae.And infer whether the bone resorption function of mature osteoclasts was inhibited.Part 3:Study on the mechanism of the effect of liensinine on bone and osteoclasts in ovariectomized mice in vitroFirstly,the osteoclast apoptosis in bone tissue was observed by TUNEL staining in the first part of the experiment.Secondly,RANKL+M-CSF was used as the induction condition in vitro to induce bone marrow mononuclear cell?BMMC?extracted from mouse bone marrow to differentiate into osteoclasts.According to the presence of inducer and liensinine,the experiment was divided into 8 groups:?1?OC?Osteoclast?+RANKL,?2?OC+RANKL+1?M Lie,?3?OC+RANKL+2?M Lie,?4?OC+RANKL+4?M Lie,?5?OC,?6?OC+1?M Lie,?7?OC+2?M Lie,?8?OC+4?M Lie.After treatment,Hoechst33258 staining was performed and osteoclast apoptosis was observed at 3 h,6 h,24 h and 3,7 days.Finally,RANKL+M-CSF were used as induction conditions to induce BMMCs to differentiate into osteoclasts,under the in vitro experimental circumstances.In this part,we set the samples into three groups.?1?blank control group,that is,uninduced differentiation group;?2?induced differentiation group;?3?induced differentiation+liensinine group?4?M?.Immunofluorescence staining?Immunofluorescence,IF?was used to detect the expression of Fas and Fas L protein,and Westernblot technique was used to detect the expression of apoptosis-related pathway proteins Fas,Fas L,caspase-8,caspase-9,caspase-3,cytochrome C,Bcl-2 and Bax.Results:The results of in vivo experiment:compared with the sham-operated group,the bone loss of ovariectomized female mice was obvious after 6 weeks of culture.In detail,The H&E staining images showed an obviously decrease of the femur trabeculae number.And the the results of Micro CT scaning showed a big decrease in trabecular number?Tb.N?.Besides,the other indecations,bone mineral density?BMD?,bone surface area and total value ratio?BS/TV?,bone volume fraction?BV/TV?and also exhibited a noteworthy decline.The results of TRAP staining showed that the proportion of osteoclasts on the surface of trabeculae of metaphysis of femur increased,the shape was larger,and the number of nuclei was more.The results of ELISA detection in venous blood showed that the contents of inflammatory cytokines TNF-?and IL-6 and osteoclast markers CTX-1and TRAc P-5b raised.However,there was no significant difference in the level of osteogenesis related markers,Osteocalcin.After the intervention of intraperitoneal injection of liensinine,we could see a noteworthy raise of the bone mineral density?BMD?.Bone surface area and total value ratio?BS/TV?,bone volume fraction?BV/TV?and the number of trabeculae?Tb.N?in ovariectomized mice were also significantly higher than those in OVX group.The number of osteoclasts on the trabecular surface of bone sections stained by TRAP decreased,and showed morphological changes such as detachment from bone surface and pyknosis.The results of serological ELISA showed that the levels of inflammatory cytokines TNF-?,IL-6 and osteoclast related markers TRAc P-5b and CTX-1 were lower than those in OVX group.The results of in vitro experiment:firstly,liensinine significantly reduced the survival number of osteoclasts in a dose-dependent manner,showing that the number of positive cells stained by TRAP was significantly less than that in the induced differentiation group.Secondly,the bone resorption function of osteoclasts was significantly inhibited by liensinine.The number and area of actin rings in mature osteoclasts under the intervention of liensinine were significantly lower than those in the induced differentiation group,and the absorptive capacity of osteoclasts to hydroxyapatite bone plate in the induced differentiation group was lower than that in the induced differentiation group.the area of bone lacuna was significantly decreased compared with induced differentiation.Conclusion:1.In vivo experiments showed that Liensinine could reduce bone loss in ovariectomized mice;reduce the number of osteoclasts on the bone surface;reduce inflammatory cytokines and osteoclast markers in ovariectomized mice.2.In vitro experiments showed that Liensinine reduce the number of osteoclasts,inhibit the bone resorption function of osteoclasts,promoted the apoptosis of mature osteoclasts induced by RANKL,and inhibit the formation of osteoclast actin ring and the bone resorption function of osteoclasts.3.Mechanism experiments revealed that liensinine enhanced the sensitivity of osteoclasts to endogenous mitochondrial apoptosis mediated by Fas/Fas L by increasing the expression of Fas and Fas L in osteoclasts,thus promoting osteoclast apoptosis in advance and shortening its life cycle,thus inhibiting osteoclast bone resorption and alleviating bone loss in ovariectomized mice.