| Research objective: Kidney deficiency and blood stasis are the common pathogenesis of osteoporosis,the prescription ofBuShen YiQi HuoXue Recipe has definite clinical effect.Based on this study Wnt3a/?-catenin signaling pathway and bone marrow mesenchymal stem cells(BMSCs)proliferation osteogenetic differentiation,explore the BuShen YiQi HuoXue Recipe action mechanism of prevention and treatment of postmenopausal osteoporosis,for "the kidney stores essence" visceral manifestation theory and application to provide some experimental basis,for the clinical prevention and treatment of postmenopausal osteoporosis has certain theoretical significance and application value.Materials and methods: Part ?: theoretical research By analyzing ancient and modern literature and retrieve the modern clinical research data,using theoretical analysis method,related to traditional Chinese medicine for osteoporosis disease,etiology,pathogenesis,syndrome differentiation and treatment of induction and summary,the key pathogenesis of osteoporosis in in-depth discussion and analysis,interpretation the profound connotation of osteoporosis in disease,etiology,pathogenesis,syndrome,treatment.Part ?: in vivo study 90 SPF SD female rats were randomly divided into 5 groups: normal group,model group,low dose group of BuShen YiQi HuoXue Recipe,high dose group of BuShen YiQi HuoXue Recipe,and fosamax group(positive drug control group).Except the normal group,the other groups were operated to remove bilateral ovaries to establish the rat model of postmenopausal osteoporosis.Normal group and model group were given normal saline by gavage,and the other groups were given corresponding volume of experimental drugs by gavage.Each group was given medicine on the second day after surgery.Daily am9:00 on time for 12 weeks.After the last intragastric administration,animals were starved for 24 h and sacrificed,corresponding indicators were detected by sampling.Wnt3 a of bone and kidney tissue of rats,CyclinD1 mRNA andprotein expressions in each group were detected by qRT-PCR and ELISA.Part ?: in vitro experiments Source of experimental cells: OriCellTMWistar rat bone marrow mesenchymal stem cells were purchased from saiye(suzhou)biotechnology co.LTD.Cell resuscitation and culture were performed according to OriCellTMWistar rat bone marrow mesenchymal stem cells instructions.Drugs used in the experiment: the effective components of BuShen YiQi HuoXue Recipe were divided into tonifying kidney group,invigorating qi and activating blood circulation group and tonifying kidney,invigorating qi and activating blood circulation group according to the functional compatibility.The aim of experiment 3 was to screen the optimal dose-response of functional compatibility groups in promoting BMSCs proliferation and osteogenic differentiation.The experiment was divided into 11 groups: normal group;Osteogenic induction group;High dose group of tonifying kidney(1);Medium dose group of tonifying kidney(2);Low dose group of tonifying kidney(3);High dose group of invigorating qi and activating blood circulation(4);Medium dose group of invigorating qi and activating blood circulation(5);Low dose group of invigorating qi and activating blood circulation(6);High dose group of tonifying kidney,invigorating qi and activating blood circulation(7);Medium dose group of tonifying kidney,invigorating qi and activating blood circulation(8);Low dose group of tonifying kidney,invigorating qi and activating blood circulation(9).Separately acting on BMSCs.Experiment 4 and 5 observed the mechanism of BMSCs proliferation and osteoblastic differentiation in each group with optimal dose functional compatibility through Wnt3a/?-catenin signal pathway related indicators.Wnt pathway inhibitor is DKK1.It was divided into 9 groups: normal group,osteogenic induction group,and medium dose group of tonifying kidney(1);Medium dose group of invigorating qi and activating blood circulation(2);Medium dose group of tonifying kidney,invigorating qi and activating blood circulation(3);Osteoblastic induction +DKK1 group(4);Medium dose of tonifying kidney +DKK1 group(5);Medium dose of invigorating qi and activating blood circulation +DKK1 group(6);Medium dose of tonifying kidney,invigorating qi and activating blood circulation +DKK1 group(7).Experimental index detection: Experiment 3: MTT assay was used to detect the proliferation of BMSCs24,48 and 72 h in each group.The ALP and BGP levels at 12 d and 15 d were detected by ELISA.Alizarin red staining was used to observe bone calcium nodules at 15 d.Experiment 4: ELISA method was used to detect the changes of ALP and BGP levels at 15 d in each group(DKK1).The bone calcium nodules were observed by alizarin red staining.The expressions of 15dWnt3 a and CyclinD1 mRNA in each group were detected by qRT-PCR.Wnt3 a protein expression in each group was detected by ELISA.SPSS17.0 statistical software was used to process the experimental data.All the data were expressed as(?)ąs,and the data in vivo and in vitro were statistically analyzed by one-way anova.P < 0.05 was statistically significant.Results: Part ?: theoretical research Osteoporosis mostly belongs to the category of "bone impotence","bone blight" and "bone pain" in traditional Chinese medicine.Kidney deficiency and pith loss is the basic pathogenesis of osteoporosis.Weak spleen and stomach,lack of qi and blood biochemical source;Or spleen deficiency and kidney,less than the exchange of blood;Liver loss,loss of qi and blood or poor operation;Or deficiency of liver and kidney,lack of blood supply;Chronic disease and kidney,multi-deficiency and multi-stasis,kidney deficiency and blood stasis;Is the common pathogenesis of osteoporosis;Most of the external factors are inducing factors.Osteoporosis fractures are mostly qi stagnation and blood stasis.Modern expert consensus divides osteoporosis into six syndromes of kidney deficiency and blood stasis,liver and kidney Yin deficiency,qi stagnation and blood stasis,kidney Yang deficiency,spleen and stomach weakness,spleen and kidney Yang deficiency.For the prevention and treatment of osteoporosis with kidney deficiency and blood stasis syndrome,it is often effective to add the product of invigorating qi and activating blood circulation into the prescription of tonifying kidney.Therefore,this study is based on the BuShen YiQi HuoXue Recipe,to carry out the experiment and research,in part to elucidate the mechanism of action.Part ?: in vivo study 1.Wnt3 a mRNA expression and protein expression changes in bone tissues of rats in each group Compared with the normal group,Wnt3 a mRNA expression of bone tissue in the model group was significantly decreased(P < 0.01).Compared with the model group,Wnt3 a mRNA expression in bone tissue of each treatment group was up-regulated to varying degrees(P < 0.01),and the most obvious was that in the fosamax group(P < 0.01).Wnt3 a mRNA expression in bone tissues was up-regulated in both the fosamax group and the high dose group of tonifying kidney,invigorating qi and activating blood circulation,but the comparison between the two groups was not statistically significant(P > 0.05).Compared with the normal group,Wnt3 a protein expression in bone tissue was significantly decreased in the model group(P < 0.01).Compared with the model group,Wnt3 a protein expression in bone tissue of each treatment group was up-regulated to varying degrees(P < 0.01),especially in the high dose group of BuShen YiQi HuoXue Recipe(P < 0.01).Wnt3 a protein expression in bone tissue was up-regulated in both the high dose group of tonifying kidney,invigorating qi and activating blood circulation and the fosamax group,but the comparison between the two groups was not statistically significant(P > 0.05).2.Changes of CyclinD1 mRNA expression in bone tissues of rats in each group Compared with the normal group,the expression of CyclinD1 mRNA in bone tissue of the model group was significantly decreased(P < 0.01).Compared with the model group,the expression of CyclinD1 mRNA in bone tissues of each group was up-regulated to different degrees(P < 0.01),and the most obvious expression was found in the fosamax group(P < 0.01).The expression of CyclinD1 mRNA in bone tissues was up-regulated in both the fosamax group and the high dose group of tonifying kidney,invigorating qi and activating blood circulation,but there was no statistical significance between the two groups(P > 0.05).3.Wnt3 a mRNA expression and protein expression changes in rat kidney tissues of each group Compared with the normal group,the expression of Wnt3 a mRNA in renal tissue of the model group was significantly decreased(P < 0.01).Compared with the model group,the expression of Wnt3 a mRNA in renal tissues of each treatment group was up-regulated to varying degrees(P < 0.01),especially in the fosamax group(P < 0.01).Wnt3 a mRNA expression in bone tissue was up-regulated in both the fosamax group and the high dose group of tonifying kidney,invigorating qi and activating blood circulation,but there was no statistical significance between the two groups(P > 0.05).Compared with the normal group,Wnt3 a protein expression in renal tissue of the model group was significantly decreased(P < 0.01).Compared with the model group,the expression of Wnt3 a protein in kidney tissues of the other groups was up-regulated to varying degrees(P < 0.01),especially in the high dose group of BuShen YiQi HuoXue Recipe(P < 0.01).Next was the fosamax group.4.Changes of CyclinD1 mRNA expression in renal tissues of rats in each group Compared with the normal group,the expression of CyclinD1 mRNA in renal tissues of the model group was significantly decreased(P < 0.01).Compared with the model group,the expression of CyclinD1 mRNA in renal tissues of the other groups was up-regulated to varying degrees(P < 0.01),and the most obvious expression was found in the fosamax group(P < 0.01).The expression of CyclinD1 mRNA in renal tissues was up-regulated in both the high dose group of tonifying kidney,invigorating qi and activating blood circulation,but there was no statistical significance between the two groups(P > 0.05).Part ?: in vitro experiments 1.Proliferation of BMSCs in each group at 24 h,48h and 72h(experiment 3)24h: compared with the osteoblastic induction group,there was no significant difference in the proliferation of BMSCs in the high dose group of tonifying kidney(P > 0.05),There was no significant difference in BMSCs proliferation in the medium dose group of tonifying kidney,and the medium dose group of tonifying kidney,invigorating qi and activating blood circulation(P > 0.05).The proliferation of BMSCs in other groups decreased,but there was no statistical significance(P > 0.05).48h: compared with osteogenesis induction group,high dose group of tonifying kidney BMSCs proliferation ability raise no obvious difference(P > 0.05),medium dose group of invigorating qi and activating blood circulation,medium dose group of tonifying kidney,invigorating qi and activating blood circulation BMSCs proliferation close,no statistical significance(P > 0.05),and other drug group of BMSCs proliferation decreases,but without statistical significance(P > 0.05).72h: compared with the osteoblastic induction group,the BMSCs proliferation capacity of the high dose group of tonifying kidney,the low dose group of invigorating qi and activating blood circulation and the medium dose group of tonifying kidney,invigorating qi and activating blood circulation was similar,without statistical significance(P > 0.05),and the BMSCs proliferation of the other groups was decreased,but without statistical significance(P > 0.05).2.Changes in ALP levels at 12 d and 15 d in each group(experiment 3)12d: compared with the osteoblastic induction group,ALP levels in the high,medium and low dose groups of tonifying kidney,the high,medium and low dose groups of invigorating qi and activating blood circulation,and Low dose group of tonifying kidney,invigorating qi and activating blood circulation were decreased(P < 0.01),while ALP levels in the high and medium dose groups of tonifying kidney,invigorating qi and activating blood circulation were close to those in the osteoblastic induction group(P > 0.05).15d: compared with the osteoblastic induction group,the ALP level in each group decreased(P < 0.01).Among them,the ALP level was slightly higher in the medium dose group of tonifying kidney,the medium dose group of invigorating qi and activating blood circulation,the high and medium dose group of tonifying kidney,invigorating qi and activating blood circulation,but without statistical significance(P > 0.05).3.Changes of BGP level in each group at 12 d and 15d(experiment 3)12d: compared with the osteogenic induction group,the BGP level of each group was decreased(P < 0.01),among which,the BGP level was slightly higher in the medium dose group of tonifying kidney,the high,medium and low dose group of tonifying kidney,invigorating qi and activating blood circulation,but there was no statistical significance(P > 0.05).15d: compared with the osteoblastic induction group,BGP level in each group decreased(P < 0.01).Among them,BGP level was slightly higher in the high,medium and low dose group of tonifying kidney,invigorating qi and activating blood circulation,but without statistical significance(P > 0.05).4.15 d morphology of bone calcium nodules(experiment 3)Based on the observation of cell morphology of calcium bone nodules by alibi red staining,it was found that the osteogenic induction group,the medium dose group of tonifying kidney and the medium dose group of tonifying kidney,invigorating qi and activating blood circulation were the most obvious,suggesting that BMSCs could promote the formation of calcium bone nodules in the process of osteogenic differentiation.5.Changes in ALP level of cells in each group(DKK1 was added into the drug group)at 15d(experiment 4)Compared with the normal group,the ALP level in the osteogenic induction group was significantly increased(P < 0.01).The ALP level in the osteoblastic induction +DKK1 group decreased significantly(P < 0.01).Compared with the osteogenic induction group,the ALP level in the tonifying kidney group was significantly different(P < 0.01).There was a slight decrease in the group of invigorating qi and activating blood circulation and the group of tonifying kidney,invigorating qi and activating blood circulation(P < 0.01).Compared with the osteoblastic induction +DKK1 group,the ALP levels in the tonifying kidney +DKK1 group,invigorating qi and activating blood circulation +DKK1 group and tonifying kidney,invigorating qi and activating blood circulation +DKK1 group all decreased(P < 0.01),but the ALP levels in the tonifying kidney,invigorating qi and activating blood circulation group were significantly higher than those in the tonifying kidney group +DKK1 group,invigorating qi and activating blood circulation group +DKK1 group(P < 0.01).6.Changes of BGP level in each group(DKK1 was added into the drug group)at day 15(experiment 4)Compared with the normal group,the BGP level in the osteogenic induction group was significantly increased(P < 0.01).BGP level in the osteoblastic induction +DKK1 group decreased significantly(P < 0.01).Compared with the osteogenic induction group,there was no significant difference in BGP level between the tonifying kidney group and the osteogenic induction group(P > 0.05).There was a slight decrease in invigorating qi and activating blood circulation group and tonifying kidney,invigorating qi and activating blood circulation group(P > 0.05).Compared with the osteogenic induction +DKK1 group,the levels of BGP in the tonifying kidney +DKK1 group,invigorating qi and activating blood circulation +DKK1 group and tonifying kidney,invigorating qi and activating blood circulation +DKK1 group all decreased(P < 0.01),but the levels of BGP in the tonifying kidney,invigorating qi and activating blood circulation +DKK1 group were significantly higher than those in the tonifying kidney +DKK1 group and invigorating qi and activating blood circulation +DKK1 group(P < 0.01).7.Morphology of bone calcium nodules at 18d(DKK1 was added into the treatment group)(experiment 3)Based on the observation of cell morphology of calcium bone nodules by alibi red staining,it was found that the osteogenic induction group,the medium dose group of tonifying kidney and the medium dose group of tonifying kidney,invigorating qi and activating blood circulation were the most obvious,suggesting that BMSCs could promote the formation of calcium bone nodules in the process of osteogenic differentiation.8.Wnt3 a mRNA and protein expression in each group at 15d(experiment 4)Compared with the normal group,Wnt3 a mRNA expression was significantly up-regulated in the osteogenic induction group(P < 0.01).Wnt3 a mRNA expression was significantly decreased in the osteoblastic induction +DKK1 group(P < 0.01).Compared with the osteogenic induction group,the expression of Wnt3 a mRNA in the tonifying kidney group and invigorating qi and activating blood circulation group was significantly decreased(P < 0.01),while that in the tonifying kidney,invigorating qi and activating blood circulation group was up-regulated(P < 0.01).Compared with the osteoblastic induction group +DKK1 group,the expression of Wnt3 a mRNA in the tonifying kidney,invigorating qi and activating blood circulation +DKK1 group was significantly up-regulated(P < 0.01).tonifying Kidney +DKK1 group was significantly upregulated(P < 0.01).Compared with the normal group,Wnt3 a protein expression was significantly up-regulated in the osteogenic induction group and the osteogenic induction +DKK1 group(P < 0.01).Compared with the osteogenic induction group,Wnt3 a protein expression was decreased in the tonifying kidney group,invigorating qi and activating blood circulation group and tonifying kidney,invigorating qi and activating blood circulation group(P < 0.01).Compared with the osteoblastic induction +DKK1 group,there was no significant difference in the expression of Wnt3 a protein in the tonifying kidney,invigorating qi and activating blood circulation +DKK1 group(P > 0.05),while the expression of Wnt3 a protein in the invigorating qi and activating blood circulation +DKK1 group was significantly reduced(P < 0.01).9.Expression of CyclinD1 mRNA in each group at 15d(experiment 4)Compared with the normal group,the osteogenic induction group was significantly upregulated(P < 0.01).Compared with the osteogenic induction group,the expression of CyclinD1 mRNA in the tonifying kidney,invigorating qi and activating blood circulation group was significantly up-regulated(P < 0.01),while the expression of CyclinD1 mRNA in the tonifying kidney group and invigorating qi and activating blood circulation group was decreased(P < 0.05).Compared with the osteoblastic induction +DKK1 group,the expression of CyclinD1 mRNA in the tonifying kidney,invigorating qi and activating blood circulation +DKK1 group was significantly up-regulated(P < 0.01),followed by the tonifying kidney +DKK1 group(P < 0.05),and there was no significant difference between the two groups(P > 0.05).Conclusions: 1.Osteoporosis belongs to the category of "bone impotence","bone deficiency" and "bone pain" in traditional Chinese medicine.The deficiency of kidney marrow is the basic pathogenesis of osteoporosis.Weakness of spleen and stomach,deficiency of liver and kidney,deficiency of spleen and kidney,deficiency of kidney and blood stasis are common pathogenesis of osteoporosis.Therefore,most of the clinical treatments are tonifying kidney and marrow,liver and kidney,spleen and blood,and qi and blood.2.BuShen YiQi HuoXue Recipe can up-regulate the expressions of Wnt3 a,CyclinD1mRNA and Wnt3 a protein in bone and kidney tissues of postmenopausal osteoporosis model rats,thus playing a role in the prevention and treatment of osteoporosis.3.The effective components of BuShen YiQi HuoXue Recipe were compatible with each other.Each group of kidney-tonifying,qi-activating and blood-tonifying prescription could promote the proliferation and osteogenic differentiation of BMSCs.The most effective components of kidney-tonifying and qi-activating prescription could increase the level of ALP and BGP and promote the formation of calcium bone nodules.4.BMSCs osteogenic differentiation was related to the expression of Wnt3 a mRNA,the upstream promoter protein,and CyclinD1 mRNA in the Wnt cell pathway.Under the effect of Wnt pathway inhibitor DKK1,the effective components of BuShen YiQi HuoXue Recipe can activate the inhibition of Wnt signaling pathway and effectively up-regulate the mRNA and protein expression of Wnt3 a,the upstream promoter protein of Wnt3a/?-catenin signaling pathway,and the expression of CyclinD1 mRNA,the downstream related indicator,so as to play a role in the prevention and treatment of osteoporosis.5.BuShen YiQi HuoXue Recipe and its effective components can play a role in prevention and treatment of osteoporosis by regulating Wnt3a/?-catenin signal pathway. |
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