| Osteoporosis?OP?is a systemic skeletal disease,mainly characterized by lowering bone mass and bone weight per unit volume generating in sufficient organics,and there by decreasing calcium deposition.It showed that the bone epiphyses became thinner and porous,the trabecular cracks were more numerous and the net-structure was destroyed.As a result,bone strength was decreased,brittleness was increased,and fracture was easily occurred.Osteoporosis has become an important metabolic disease that affects health all over the world.In modern medicine,osteoporosis is divided into primary and secondary categories.Primary osteoporosis is divided into type I and type II.Postmenopausal osteoporosis?PMOP?belongs totype I.Estrogen deficiency which occurs to menopause plays a major role in the development of osteoporosis in post-menopausal women.In order to prevent adverse effects of estrogen deficiency in bone,estrogen replacement therapy has been proposed.However,long-term use of estrogen may increase endometrial cancer and breast cancer risk.Many investigators have shown that a class of plant-derived substances had estrogenic activities,they are so-called“phytoestrogen”.These include:flavonoid,coumarin,lignans etc.Schizandrin is a lignan-class derivative extracted from Chinese herbs.Schisandra chinensis have been applied to treat bone diseases for thousands of years.However,the role of Schizandrin in osteoblast function and the underlying molecular mechanisms remain poorly understood.Part 1:Effects of Schizandrin on the proliferation and differentiation of rat osteoblastsObjective:The effects of Schizandrin on the proliferation and differentiation of rat osteoblasts.Method:1.Primary cultured osteoblasts and observation of their morphology.2.MTT assay.3.ALP activity assay.Results:1.Morphological observation and identificationUnder inverted microscope,the osteoblast which is inoculated is globular at first and then suspend in culture fluid.After 24 hours,the cells attached to the wall and showed triangle or polygonal in the early stage,then they became fusiform,elongated.After 72 hours,cell density increased visibly,the elongated cells become oval,and the boundary between the cells gradually reduced.After the alkaline phosphatase staining assay,cells's nucleolu turned blue and the active site of the enzyme turned brownish black.2.MTT assayAfter being cultivated 36 h,compared with control group,the concentration of 1.0×10-55 mol/L and 1.0×10-6 mol/L could promote the proliferation of osteoblast.After being cultivated 48 h,1.0×10-55 mol/L1.0×10-77 mol/L could promote the proliferation of osteoblast.3.ALP activity assayCompared with control group,after being cultivated 48 h and 72 h,the concentration of 1.0×10-55 mol/L and 1.0×10-66 mol/L could promote the differentiation in two periods.Conclusions:1.Schizandrin had significant effects on osteoblast growth.2.Schizandrin could enhance the ALP activity.Part 2:Schizandrin stimulates rat osteoblasts differentiation through activation of BMP-Smads signal pathwayObjective:Studies have shown that many signaling pathways are involved in the differentiation of osteoblasts,and BMP-Smads signal transduction pathway is one of the major pathways for osteoblast differentiation.In this part,we studies the stimulatory effect of Schizandrin on differentiation in rat osteoblasts through BMP-Smads signal pathway.Method:1.Primary cultured osteoblasts and observation of their morphology.2.qPCR assay.3.Western blot assay.Results:1.Morphological observation and identificationThe results of osteoblasts culture and identification are similar to the first part.2.qPCR assayCompared with control group,after being cultivated 48 h,72 h,The concentration of 1.0×10-55 mol/L and 1.0×10-66 mol/L could promote the Osx gene mRNA espreeion level.3.Western blot assayAfter being cultivated 2 h,the p-Smad1/9 protein expression peaked.After cultivated 12 h,Smad1/5/9 protein expression had a rising trend.After being cultivated 48h,1.0×10-66 mol/L could express the highest Osx protein level.Conclusions:1.Compared with control group,Osterix mRNA expression were rised.2.Compared with control group,Smad1/5/9 protein,p-Smad1/9 protein,Osx protein level were risedPart 3:Effects of Schizandrin respectively combined with Isopsoralen and kaempferol on the proliferation and differentiation of rat osteoblastsObjective:Traditional Chinese medicine formulas have a long history of use in the prevention and treatment of osteoporosis.Schisandra chinensis often appeared in the formulas with fructus psoraleae or Semen cuscutae.This study observed the effect of Schizandrin combined with isopsoralen or kaempferol on proliferation and differentiation of rat osteoblasts.Method:1.Primary cultured osteoblasts and observation of their morphology.2.MTT assay.3.ALP activity assay.Results:1.Morphological observation and identificationThe results of osteoblast culture and identification are the same as the first part.2.MTT assayCompared with control group and the single compound group,two drugs together could promote the effects of proliferation.3.ALP activity assayCompared with control group and the single compound group,two drugs together could enhance the ALP activity.Conclusions:Compared with control group and the single compound group,Schizandrin respectively with Isopsoralen and kaempferol could promote the proliferation and ALP activity of ostesblasts.Schizandrin can promote the proliferation and differentiation of osteoblast.It might promote osteoblast differentiation by BMP2-Smads signaling pathway. |
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