Anti-osteoporosis Effect Of Schisandrin

[Objective]:Osteoporosis is a systemic bone disease caused by multiple factors that lead to decreased bone mineral density and bone mass,destruction of bone micro structure,and increased bone fragility,which can easily lead to fracture.It's common in the elderly and postmenopausal women.When women are in menopause period,their estrogen withdrawal is reduced,causing progressive bone loss.The incidence of postmenopausal osteoporosis(PMOP)in postmenopausal women is as high as 50%.The sixth national census in China shows that about 54.10 million women over age 50 suffer from osteoporosis.Estrogen replacement therapy(ERT)can lead to inhibition of bone loss and is currently the preferred method for treating PMOP.Estrogen-treated patients have a 30%increased risk of breast cancer,and the use of estrogen alone may increase the risk of endometrial cancer.Choosing an ideal estrogen replacement medicine which is not only preserving the anti-osteoporosis effect of estrogen,but also avoiding the side effects of estrogen on sexual organs is of great significance.Compounding with a similar estrogenic structure and a weak estrogen-like effect from plants are called phytoestrogen(PE).Its mechanism of action has a very large relationship with estrogen levels in the body.For low estrogen levels,it can be combined with estrogen receptors to show estrogen-like effects;for high estrogen levels,it can compete with estrogen for estrogens.Hormone receptors thus exhibit anti-estrogen effects.Studies have shown that phytoestrogens such as soy isoflavones and epimedium have anti-osteoporosis effects and are much lighter in side effects than estrogens.However,due to soybeans belonging to beans,allergies related to allergy to beans may appear.Excessive use of isoflavones has been found to cause iodine deficiency and lead to a decline in thyroid function,which may be related to the potential toxicity of isoflavones as substrates for thyroid peroxidase.Schisandra is a widely used traditional Chinese medicine that is used for calming and nourishing.Lignans,volatile oils,organic acids,polysaccharides and other chemical component form Schisandra.Lignin is its most important active ingredient,its composition contains biphenyl cyclooctene skeleton,its pharmacological effects involving digestion,cardiovascular,central nervous system,reproductive and urinary systems.In the past,the physiological activity of lignans was mostly used to protect the liver from down-regulating enzymes and anti-tumor effects.Now its anti-osteoporosis effect is getting more and more attention.Schisandrin(SCH)(molecular formula:C24H3207)is a very important active monomer in Schisandra,and its role in anti-osteoporosis has not been reported.In view of this,this paper studies the anti-osteoporosis effect of schisandrin and its mechanism of action,providing the theoretical basis for basic research.[Mathods]:Through the combination of ex vivo cell culture and animal experiments,the effect and mechanism of Schisandrin against osteoporosis were explored.In the cell experiment section,different concentrations of Schisandrin(0 nmol/L,1 nmol/L,10 nmol/L,100 nmol/L,1?mol/L,10?mol/L)were added to the mouse osteoblast culture medium.The MTT assay was used to test the effect of Schisandrin on the proliferation and differentiation of osteoblasts.The change in alkaline phosphatase(ALP)activity in cells was determined by a kinetic assay of nitrobenzene disodium matrix.OCN was measured by RTPCR.Collagen type I collagen measured by western blot.Osteoblasts was stained with alizarin red to test the mineralization.The expression of Runx-2 was measured using western blot.After adding estrogen receptor inhibitor ICI182780(ICI),100nmol/L SCH,and ICI+SCH for 72 hours,the ALP activity of osteoblast was measured,the mRNA expression of COL-1 was measured by RTPCR,and the expression of Runx-2 was detected by western blot.After osteoblasts were cultured,100nmol/L SCH was added,and Western blot was used to measure ERK,p38,JNK and their phosphorylated proteins expressions at 0min 10min 20min 30min 40min 60mint.After osteoblasts were cultured and ICI,SCH,ICI+SCH were added,the expression of ERK,p38,JNK,and their phosphorylated proteins were measured by western blot.The three inhibitors of MAPK,SB203580,SP600125,and PD184352(respectively inhibiting the phosphorylation of P38,JNK,and ERK,respectively)were added to measure ALP activity and COL-1 mRNA expression in osteoblasts.In the animal experiments section,a mouse model of osteoporosis was established by removing bilateral ovaries.Group feeding:sham-operated groups(removal of bilateral parafacial fat);surgical groups(removal of bilateral ovaries);removal of bilateral ovarian surgery plus low dose(20 mg/kg)SCH gavage qd*10 weeks;removal of bilateral ovarian surgery plus high dose(40mg/kg)SCH gavage qd *10 weeks.All mice were ocularly bled and sacrificed after 10 weeks of feeding.Detect tartrate-resistant acid phosphatase(TRACP)and bone alkaline phosphatase(BLAP).The femoral muscle of the hindlimb was excised and the proximal femoral bone density was measured by microCT.The bone tissue RNA was extracted by one-step method of isothiocyanate and chloroform,and the COL-1 mRNA content was detected by RTPCR.[Results]:In cytology experiments,MTT assay result showed that various concentrations SCH significantly promoted proliferation of osteoblasts at 24h,48h,and 72h.When osteoblasts were cultured for 72 h and 120 h at different concentrations(1×10-9 mol/L to 1×10-5 mol/L)of SCH,compared with the control group(0 concentration group),each concentration of SCH showed a promoting effect on the ALP activity of mouse osteoblasts.Realtime-PCR results showed that after treatment of mouse osteoblasts with SCH from1×10-9 mol/L to 1×10-5 mol/L for 72 h,the OCN mRNA expression level was significantly different to that of the control group without SCH.The maximum effective concentration was 1×10-7 mol/L.Western blot analysis showed that after osteoblasts were treated with SCH for 72 h,compared with the control group without SCH,the level of COL-1 protein was significantly increased.Alizarin red staining showed that mouse osteoblasts with or without 100 nm SCH for 21 days were significant differences in mineralized nodules of osteoblasts.After the addition of estrogen receptor inhibitor ICI182780,the activity of ALP in osteoblasts of mice treated with schisandrol was significantly lower than that without inhibitor(P<0.01);the expression of COL-1 mRNA in mouse osteoblasts was inhibited.The agent was significantly reduced(P<0.01).Western blot analysis showed that when mice osteoblasts were treated with 1×10-9 mol/L to 1×10-5 mol/L SCH for 72 h,compared with the control group without SCH,the level of Runx2 protein increased at SCH concentrations of 1×10-6 mol/L and 1×10-7 mol/L(P<0.05).After the addition of ICI,the protein expression of Runx2 in mice osteoblasts was significantly lower than that of the inhibitor ICI182780(P<0.05).After 100nmol/L SCH added to mice osteoblasts,the protein expression of ERK,P38,JNK,and their phosphorylation was measured by western at 0min 10min 20min 30min 40min 60mint.The expression of ERK,P38,and JNK protein was not significantly increased with time(P<0.05),and the expression of p-ERK,p-JNK,and p-P38 protein was significantly increased(P<0.01).After the addition of estrogen receptor inhibitor ICI182780,the promoting effect of SCH on mice osteoblast MAPK signaling pathway protein expression was inhibited(P<0.01).After the addition of MAPK phosphorylation inhibitors PD,SP,SB,the ALP activity with SCH and the promotion of COL-1 mRNA expression were inhibited(P<0.01).In animal experiments,after 10 weeks of treatment with SCH,the bone density of ovariectomized mice increased(P<0.05),and type I collagen in femurs increased(P<0.01).BALP and TRACP are markers of bone formation and bone resorption,respectively,and are reduced after treatment with SCH.[Conclusion]:As a phytoestrogen,SCH has an effect of promoting proliferation of mice osteoblasts and has the greatest effect at a concentration of 1×10-7 mol/L.SCH can increase the activity of ALP witch is early differentiation markers of osteoblasts;SCJ can promote the secretion of type I collagen witch is the earliest differentiation marker of osteoblasts;SCH can promote OCN secretion witch is a marker of late differentiation of osteoblasts.SCH combined with estrogen receptors promotes the proliferation and differentiation of osteoblasts.Runx2 is an osteoblast-specific transcription factor that plays a major role in the formation and reconstruction of bone tissue.SCH combined with estrogen receptors promoted the generation of Runx2 in osteoblasts.The mitogen-activated protein kinase(MAPK)signal transduction pathway activated by SCH combinding estrogen receptors can promote osteoblast proliferation and differentiation.After 10 weeks of treatment with SCH,the bone density of the ovariectomized mice increased and the type I collagen of the femur increased.This shows that SCH can prevent osteoporosis.This experimental model is a high-transformation osteoporosis characterized by increased bone formation and increased bone resorption.However,bone resorption is greater than bone formation.This leads to osteoporosis.BALP and TRACP,as indicators of bone formation and bone resorption,were found decreases after treatment with SCH.This shows that SCH can prevent osteoporosis by reducing the bone turnover rate.