| Exposure to weightlessness during space flight may lead to a state of structural and functional alteration in astronauts'skeleton system,which is manifested clearly by the loss of bone mass and bone mineral,declines in bone mechanical function and negative calcium balance.This is a serious threat to the health of astronauts and successful execution of the mission.Therefore,the research of osteoporosis,especially osteoporosis caused by microgravity or mechanical inactivity and its prevention measures,has become one of the most important subjects in the field of aerospace science and aerospace medicine.Some progress has been made in the application of Chinese herbal medicine in the field of aerospace medicine.The treasure-house of traditional Chinese medicine should be further developed and utilized by the aerospace medical workers.Drynariae Rhizoma is the dried rhizome of perennial pteridophyte Drynaria fortunei?Kunze ex Mett.?J.Sm.It is officially listed in Chinese pharmacopoeia and traditionally used for the treatment of bone fractures or related diseases.However,the modern pharmacology research of Rhizoma drynariae was started just from the early nineties.Our study aimed to investigate the anti-osteoporotic effects of DRTF as well as naringin and neoeriocitrin,the two major flavonoid compounds in Drynariae Rhizoma on osteoporosis induced by hindlimb unloading?HLU?and osteoporosis induced by ovariectomy?OVX?in rat model.Furthermore,the effect of DRTF,naringin and neoeriocitrin on osteoblast MC3T3-E1 and their molecular mechanisms were explored in the research.The main research work and results obtained in present study are as follows:1.Total flavonoids of Drynariae Rhizoma prevent bones loss induced by hindlimb unloading in ratsA hindlimb unloading tail-suspended rat model?HLU?was established to determine the effect of DRTF on bone mineral density?BMD?,biomechanical strength and trabecular bone microarchitecture.Twenty-eight male Sprague–Dawley rats were divided into four groups:baseline group,control group,hindlimb unloading with vehicle?HLU?,and hindlimb unloading treated with DRTF?HLU-DRTF,75?mg/kg/d?.Oral DRTF was administrated for 28 d.The underling mechanisms of DRTF actions on disuse-induced osteoporosis were also discussed.Crompared with HLU group,DRTF treatment significantly increased the bone mineral density and mechanical strength of the tail-suspended rats.Enhanced bone turnover markers under HLU treatment were attenuated by DRTF administration.Deterioration of trabecular bone induced by HLU was prevented through elevated bone volume/tissue volume?BV/TV?,trabecular number?Tb.N?,trabecular thickness?Tb.Th?as well as decreased the trabecular separation?Tb.Sp?.In addition,the antiosteoporosis effects of DRTF on HLU rats were demonstrated probably via activation of the Wnt/?-catenin signaling.The present study provided the first evidence that DRTF prevent the bone loss induced by HLU treatment,indicating its potential application in the treatment of disuse-induced osteoporosis.2.Total flavonoids of Drynariae Rhizoma prevent bone loss induced by ovariectomized rat modelEstrogen deficiency is one of the major causes of osteoporosis in postmenopausal women.So we investigated the therapeutic effects of DRTF on estrogen deficiency-induced bone loss using an ovariectomized rat model?OVX?and osteoblast-like MC3T3-E1 cells.Our results indicated that DRTF produced osteo-protective effects on the ovariectomized rats in terms of bone loss reduction,including decreased levels of bone turnover markers,enhanced biomechanical femur strength and trabecular bone microarchitecture deterioration prevention.What's more,DRTF administration prevent estrogen deficiency induced bone loss without hyperplastic effects on the uterus and other organs.These effects seem to be related to the high content of flavonoids that behave as phytoestrogens but did not act the same as estrogen.The in vitro study using MC3T3-E1 cells provided further support for the potential action of DRTF on bone.DRTF not only promoted viability and increased ALP activity but also enhanced the formation of mineralized nodules in osteoblasts.These results indicated that DRTF might participate in multiple stages of the osteoblast differentiation process,from early to terminal stages,to stimulate osteoblast cell differentiation and maturation.3.Effect of DRTF medicated serum on osteoblast viability,differentiation and mineralizationIn this experiment,the effects of DRTF medicated serum on MC3T3-E1 cell viability,differentiation and mineralization were studied.30 SD rats were randomly divided into control group and experimental group.Each group was given the corresponding medicine?the control group was treated with the same volume of saline?.One week after administration,the rats were anesthetized and the serum was prepared.MC3T3-E1 cells were cultured with different concentration?1.25%,2.5%and 5%?of DRTF medicated serum,then the cell viability,differentiation and calcified nodule formation were detected.MTT results showed that different concentrations of DRTF medicated serum could significantly promote the viability of MC3T3-E1 cells compared with the control group.The ALP and OC in 2.5%DRTF medicated serum group were significantly higher than that of control;2.5%DRTF medicated serum group could significantly promote the formation of mineralized nodules of MC3T3-E1 cells.The results also illustrated the effectiveness of Rhizoma Drynariae by the way of oral administration.4.Effects of naringin and neoeriocitrin on viability,differentiation and mineralization of osteoblastsThe results showed that naringin at the concentration of 1×10-88 mol/L1×10-66 mol/L and neoeriocitrin at the concentration of 1×10-99 mol/L1×10-66 mol/L could significantly promote the viability of osteoblasts.Naringin at the concentration of 1×10-7 mol/L1×10-66 mol/L and neoeriocitrin at the concentration of 1×10-88 mol/L1×10-66 mol/L could significantly promote the ALP activity of osteoblast.Thus,neoeriocitrin showed a stronger effect in promoting proliferation and differentiation of osteoblasts.MC3T3-E1 cells were osteogenic induced by naringin(1×10-7 mol/L)and neoeriocitrin(1×10-7 mol/L)for 3 d,the content of OC was significantly increased by neoeriocitrin.At 9 d and 12 d,the secretion of OC in neoeriocitrin group was significantly higher than that of naringin group.Continuous treatment on osteogenic differentiation of MC3T3-E1 cells by naringin(1×10-7 mol/L)and neoeriocitrin(1×10-7 mol/L)after 12 d,the number of mineralized nodules by naringin and neoeriocitrin were significantly higher than that of control group.Both naringin and neoeriocitrin could promote the proliferation,differentiation and mineralization of osteoblasts,especially neoeriocitrin.5.Study on the mechanism of osteoblastic cells by DRTF,naringin and neoeriocitrinDRTF,naringin and neoeriocitrin significantly promote the gene expression of Frizzled?Lrp5?Lrp6?Dsh and?-catenin gene expression which belonged toWnt/?-catenin signaling pathway.Moreover,the protein expression changes of Wnt signaling pathway were tested by western blotting,The results showed that DRTF,naringin and neoeriocitrin could significantly increase the expression of?-catenin,the expression of c-myc was up-regulated by DRTF,but there was not significant difference.BMP-2 which belonged to BMP signaling pathway was significanttly up-regulated by naringin and neoeriocitrin.DRTF,naringin and neoeriocitrin,three of them had significant effects on Runx-2 and Osterix.DRTF could significantly increase the protein expression of p-ERK and p-P38,However,the effects of naringin and neoeriocitrin on p-ERK and p-P38 were more obvious than DRTF.So we hypothesized that the main effects of DRTF on p-ERK and p-P38 which belonged to MAPK signaling pathway mainly through naringin and neoeriocitrin.DRTF,naringin and neoeriocitrin up-regulate the expression of ER?and ER?in the ER signaling pathway.Among them,DRTF play a prominent role in promoting the expression of ER?.The results showed that DRTF exert estrogen-like activities on bone and might serve as a potential source of phytoestrogens for the management of PMOP.The results of OPG/RANK/RANKL signaling pathway showed that DRTF,naringin and neoeriocitrin,all three could inhibit the differentiation and maturation of osteoclasts by regulating the ratio of OPG/RANKL in osteoblasts.Naringin and neoeriocitrin could significantly up-regulate the expression of OPG in OPG/RANK/RANKL signaling pathway,while DRTF not only significantly up-regulated the expression of OPG,but also significantly down-regulated the expression of RANKL.The results demonstrated that there are other active components in DRTF coordination to play a role on the OPG/RANK/RANKL signaling pathwayAbove all,DRTF administration effectively suppress the of bone loss induced by hindlimb unloading rat model and ovariectomized rat model.In vitro experiments showed that DRTF promotes osteoblast activity and inhibits osteoclast activation through multiple signal transduction pathways.DRTF appear to have the beneficial effects on bone metabolism based on indicators of bone formation and bone resorption.In a word,Chinese medicines have the following characteristics:multiple ingredients,multiple targets and multiple pathways to be effective.It's also suggested that DRTF has the potential of further developing into an aerospace drugs for the prevention and treatment of bone loss. |
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