| Purpose To explore the effect of Bushen Yiqi Huoxue Decoction on the differentiation of bone marrow mesenchymal stem cells in rats,and to elucidate the mechanism of Bushen Yiqi Huoxue Decoction on prevention and treatment of postmenopausal osteoporosis?POP?,and to provide new ideas for clinical prevention and treatment of OP,While providing theoretical basis for the development of new drugs.Methods Part ?: Theoretical Studies This paper summarizes the etiology and pathogenesis of bone diseases such as "bone atrophy" and "bone deficiency" from the Spring and Autumn Period to the Ming and Qing dynasties,and retrieves the understanding of the etiology and pathogenesis of osteoporosis in modern literature,Explain the origin of theoretical research on osteoporosis.Part ?: In vivo Experiments Fifty-four female SD rats were randomly divided into normal group,sham operation group,model group,Bushen Yiqi Huoxue group and Fushanmei group.The rats were randomly divided into normal group,sham operation group,model group,Group,a total of 5 groups,each group of 15.The rats were treated with Bushen Yiqi Huoxue Decoction as the therapeutic drug and Fu Shanmei as the control drug.The rats were treated with DAX to detect the bone mineral density of the rats in the left hind limbs.The expression of ALP / TRACP,OPG / RANKL protein was detected by ELISA in the serum of rats in each group.The model was established to evaluate the establishment of osteoporosis rat model and the curative effect.The m RNA and protein expression of HES1,JAG1 and Notch1 in bone and kidney were detected by q RT-PCR and ELISA.The data were analyzed by SPSS-17.0.Part ?: In vitro Experiments Cell source Bone marrow mesenchymal stem cells?BMSCs?were fed from the Laboratory of Basic Medical College of Liaoning University of Traditional Chinese Medicine.Experimental drug Icariin: Lot 200415 Liaoning North Pharmaceutical Technology Development Co.,Ltd.Astragaloside ? : batch number 201314 Liaoning North Pharmaceutical Technology Development Co.,Ltd.Hydroxy safflower yellow A: lot 201308 Liaoning North Pharmaceutical Technology Development Co.,Ltd.Rehmannide A: Lot XJ0706LA14 Beijing Ying Ze Na New Chemical Technology Research Institute Velvet: deer farm in Qingyuan,Liaoning Cell culture BMSCs were resuspended in a 100 ml culture flask by pipetting.The complete culture medium?DMEM + 10 m L / d L fetal bovine serum containing 100 U / m L blue,streptomycin?was added to the cells.?80%?,with the pipette aspiration of the culture medium,adding 0.25% trypsin digestion for 3 minutes,inverted microscope to observe the cell gradually fall off,add the cell to the cell growth state,DMEM?low sugar complete medium?solution to terminate the digestion,with a pipette gently blow to the suspension state,the cell suspension into the 15 m L centrifuge tube,1200 rpm centrifugal 10 minutes,to the supernatant,add DMEM?low sugar completely Medium?to adjust the cell concentration to 1 × 105 / m L,1: 3 ratio inoculated in 25 m L cell culture flask The flask into 5% CO2,37 ? incubator,every 2d pass 1 times,about passaged 4 times after the start of the test.Experimental grouping The rat bone marrow mesenchymal stem cells were divided into the following 20 groups according to the orthogonal design of 5 factors Normal group?DMEM + 10 m L / d L fetal bovine serum containing 100 U / m L blue,streptomycin?(DMEM +10 m L / d L fetal bovine serum containing 100 U / m L green,streptomycin + 1 × 10-7 mol / L dexamethasone +50 g / L vitamin C + 10 mmol / L B-Sodium glycerophosphate)Group 1: Icariin 1 deer antler 1 astragaloside 1 hydroxyl safflower 1 Rehmannia 1 Group 2: Icariin 1 velvet 2 Astragaloside 2 Hydroxy safflower 2 Rehmannia Group 3 : Icariin 1 Velvet 3 Astragaloside 3 Hydroxy Safflower 3 Rehmannia 3 Group 4: Icariin 2 deer antler 1 astragaloside 1 hydroxyl safflower 2 Rehmannia Group 5: Icariin 2 velvet 2 Astragaloside 2 Hydroxy safflower 3 Rehmannia 3 Group 6: Icariin 2 deer antler 3 Astragaloside 3 Hydroxy safflower 1 Rehmannia 1 Group 7: Icariin 3 Velvet 1 Astragaloside 2 Hydroxy Safflower 1 Rehmannia 3 Group 8: Icariin 3 Velvet 2 Astragaloside 3 Hydroxy Safflower 2 Rehmannia 1 Group 9: Icariin 3 deer antler 3 astragaloside 1 hydroxyl safflower 3 to yellow 2 Group 10: Icariin 1 deer antler 1 astragaloside 3 hydroxyl safflower 3 to yellow 2 Group 11: Icariin 1 Velvet 2 Astragaloside 1 Hydroxyl Red Flower 1 Rehmannia 3 Group 12: Icariin 1 deer antler 3 Astragaloside 2 Hydroxy safflower 2 Rehmannia 1 Group 13: Icariin 2 velvet 1 Astragaloside 2 Hydroxy safflower 3 Rehmannia 1 Group 14: Icariin 2 velvet 2 Astragaloside 3 Hydroxy safflower 1 Rehmannia 2 Group 15: Icariin 2 deer antler 3 Astragaloside 1 Hydroxy safflower 2 Rehmannia 3 Group 16: Icariin 3 deer antler 1 Astragaloside 3 Hydroxy safflower 2 Rehmannia 3 Group 17: Icariin 3 Velvet 2 Astragaloside 1 Hydroxy Safflower 3 Rehmannia 1Group 18: Icariin 3 Velvet 3 Astragaloside 2 Hydroxy Safflower 1 Rehmannia(1: low dose 10-8 mol / L;2: middle dose 10-7mol / L;3: high dose 10-6mol / L)Index detection Experiment 4 MTT assay was used to detect the proliferation of BMSCs in 24 h,48h and 72 h.The expression of ALP protein was detected by ELISA at 6d,9d and 12 d Alizarin red staining was used to observe the mineralized nodules at 6d,9d and 12 d of each group Experiment 5 The expression of HES1,JAG1 and Notch1 m RNA was detected by q RT-PCR in 9d and 12 d The expression of HES1,JAG1 and Notch1 protein in 9MS and 12 d of BMSCs were detected by ELISA Data processing SPSS17.0 statistical software was used to process the experimental data.All the data were expressed as ± s.The data of each group were analyzed by one-way ANOVA.Orthogonal design variance analysis was used in all experimental groups.The least significant difference method?LSD?method,P <0.05 was statistically significant.Results Part ?: Theoretical Studies Chinese medicine osteoporosis is "bone atrophy","bone dry" and other areas,the pathogenesis and kidney essence deficiency,spleen and stomach loss,liver qi deficiency and blood stasis network four related,The Treatment should be due to cause,syndrome differentiation,taking into account the specimens,the use of kidney and spleen,liver and blood,blood circulation and other treatment methods,to play the overall concept of traditional Chinese medicine and syndrome differentiation.Part ?: In vivo Experiments 1.Effect of Bushen Yiqi Huoxue Decoction on Bone Mineral Density in Ovariectomized Osteoporosis Rats Compared with the normal group,the femoral bone mineral density of the left hind limb was significantly decreased?P> 0.01?,and the density of the sham operation group did not change significantly?P> 0.05?.Compared with the model group,the bone mineral density of each treatment group increased to different degrees?P <0.01?.Among them,the bone mineral density of Fushanmei group was the most obvious,followed by Kidney Yiqihuoxue group?P <0.05?.2.Effects of Bushen Yiqi Huoxue Decoction on Bone Formation and Bone Absorption in Ovariectomized Osteoporosis Rats Compared with the normal group,the serum ALP level in the sham operation group was not significantly different?P> 0.05?,and the serum ALP content in the model group was significantly decreased?P <0.01?.Compared with the model group,the serum ALP levels of each group were significantly increased?P <0.01?,among which Fushanmei group was better than that of Bushen Chinese medicine group?P <0.01?.Compared with the normal group,the serum TRACP level in the sham operation group did not change significantly?P> 0.05?,The serum TRACP level in the blank group was significantly higher than that in the model group?P <0.01?.Compared with the model group,the serum TRACP content of each group was significantly lower?P <0.01?,There was no significant difference in TRACP level between Fushanmei group and Bushen Yiqi Huoxue group?P> 0.05?.3.Effect of Bushen Yiqi Huoxue Recipe on Expression of Serum OPG / RANKL Protein in Ovariectomized Osteoporosis Model Rats Compared with the normal group,the OPG of the sham operation group was not significantly different?P> 0.05?,and the serum OPG of the model group was significantly decreased?P <0.01?.Compared with the model group,the serum OPG ofall the drug groups increased significantly?P <0.01?,There was no significant difference in serum OPG between Fushanmei group and Bushen Yiqi Huoxue group?P> 0.05?.Compared with the normal group,the serum RANKL of the sham operation group was not significantly different?P> 0.05?,and the serum RANKL was significantly increased in the model group?P <0.01?.Compared with the model group,the serum RANKL of each group was significantly lower?P <0.01?,There was no significant difference in serum RANKL between Fushanmei group and Bushen Yiqi Huoxue group?P> 0.05?.4.Effects of Bushen Yiqi Huoxue Recipe on Expression of HES1 m RNA and Protein in Bone Tissue and Renal Tissue of Ovariectomized Osteoporosis Rats Bone tissue: Compared with the normal group,the expression of HES1 m RNA and protein in the model group was significantly decreased?P <0.01?.Compared with the model group,the expression of HES1 m RNA and protein in each drug group were significantly increased?P <0.01?,There was no significant difference in HES1 m RNA between Fu Shanmei group and Bushen Yiqihuoxue group?P> 0.05?,The expression of HES1 protein was better than that of Bushen Yiqihuoxue group?P <0.01?.The expression of HES1 m RNA and protein in the model group was significantly lower than that in the normal group?P <0.01?.Compared with the model group,the expression of HES1 m RNA and protein was significantly increased?P <0.01?.There was no significant difference in HES1 m RNA and protein expression between Fushanmei group and Bushen Yiqihuoxue group?P> 0.05?.5.Effects of Bushen Yiqi Huoxue Recipe on Expression of JAG1 m RNA and Protein in Bone Tissue and Renal Tissue of Ovariectomized Osteoporosis Rats Bone tissue: Compared with the normal group,the JAG1 m RNA content in the model group was significantly decreased?P <0.01?.Compared with the sham group,the expression of JAG1 m RNA and protein in the model group was significantly decreased?P <0.01?.Compared with the model group,the expression of JAG1 m RNA and protein increased?P <0.01?,The expression of JAG1 m RNA and protein in Fushanmei group was higher than that in Bushen Yiqihuoxue group?P <0.01?.The expression of JAG1 m RNA and protein in the model group was significantly lower than that in the normal group?P <0.01?.Compared with the model group,the expression of JAG1 m RNA and protein in each drug group was significantly increased?P <0.01?.There was no significant difference in the relative expression of JAG1 m RNA between Fushanmei group and Bushen Yiqihuoxue group?P> 0.05?,The expression of JAG1 protein in Fushanmei group was higher than that in Bushen Yiqihuoxue group?P <0.01?.6.Effects of Bushen Yiqi Huoxue Decoction on Expression of Notch1 m RNA and Protein in Bone Tissue of Ovariectomized Osteoporosis Rats Bone tissue: Compared with the normal group,the expression of Notch1 m RNA and protein in the model group was significantly decreased?P <0.01?.Compared with the blank group,the expression of Notch1 m RNA and protein in each drug group was significantly increased?P <0.01?.The expression of Notch1 m RNA in Fushanmei group was higher than that in Bushen Yiqihuoxue group?P <0.01?.There was no significant difference in Notch1 protein expression between the two groups?P> 0.05?.The expression of Notch1 m RNA and protein in the model group was significantly lower than that in the normal group?P <0.01?.Compared with the blank group,the expression of Notch1 m RNA and protein in each drug group were significantly increased?P <0.01?.There was no significant difference in the expression of Notch1 m RNA and protein between Fu Shanmei group and Bushen Yiqihuoxue group?P> 0.05?.The content of Notch1 protein in Fu Shanmei group was higher than that in Bushen Yiqihuoxue group?P <0.01?.Part ?: In vitro Experiments 1.Effect of Orthogonal Component Compatibility of Bushen Yiqi Huoxue Recipe on Proliferation of BMSCs There was no significant difference in BMSCs proliferation between the 1,3,4,7,8,9,10,11,13 groups compared with the normal group?P <0.05?The proliferation of the other groups increased significantly?P <0.01?.There was significant difference between the two groups?P <0.01?.The proliferation of BMSCs in group 18 was significantly higher than that in other groups?P <0.01?.There was no significant difference in proliferation of BMSCs between the 1,3,5,7,8,9 groups?P> 0.05?.The proliferation of the other groups increased significantly?P <0.01?.there was no significant difference between the 17 th and 18 th groups?P> 0.05?,There were significant differences among the other groups?P <0.01?.The proliferation of BMSCs in group 17 and 18 was significantly higher than that in other groups?P <0.01?.Compared with the normal group,the proliferation of BMSCs in the 15 th,16th and 17 th groups was not significantly different from that in the normal group?P> 0.05?,The proliferation of the other groups was significantly different?P <0.01?.There was no significant difference between the 10 th,12th,13 th,15th,16 th and 17 th groups?P> 0.05?,There were significant differences among the other groups?P <0.01?.The proliferation of BMSCs in the 16 th,17th and 18 th groups was significantly higher than that in the other groups?P <0.01?.The normal group,the transformant group and the 16,17,18 group reached the peak of proliferation at 48 h?24h vs 48 h P <0.01,48 h vs 72 h P <0.01?.2.Effect of orthodontic component of Bushen Yiqi Huoxue Recipe on osteogenic differentiation of BMSCs Each group of cells 6d ALP: Compared with the normal group,there was no significant difference between the first,second,third and fifth groups?P> 0.05?,he expression of ALP in the other groups was significantly increased?P <0.01?.There wassignificant difference between the two groups?P <0.01?.The expression of ALP protein in the 16 th and 18 th groups was the highest?P <0.01?.Each group of cells 9d ALP: Compared with the normal group,the activity of ALP protein was significantly increased?P <0.01?.There was significant difference between the two groups?P <0.01?.The expression of ALP protein in the 16 th and 18 th groups was the highest?P <0.01?.Each group of cells 12 d ALP:Compared with the normal group,there was no significant change in group 2?P> 0.01?,The ALP protein activity of the other groups was significantly increased?P <0.01?.Compared with the treatment group,there was no significant difference between the 16 th and 18 th groups?P> 0.05?,There were significant differences among the other groups?P <0.01?.The expression of ALP protein in the 16 th and 18 th groups was higher than that in the other groups?P <0.01?.The expression of ALP in the normal group,the transformant group and the 16 th and 18 th group were higher than those in the 6d?P <0.01?.The expression of ALP in normal group,transforming group and 16 and 18 groups was higher than that in 12 days?P <0.01?.3.Effects of Bushen Yiqi Huoxue Recipe on Expression of HES1 m RNA and Protein in 9 and 12 d of Cells in Different Groups 9d: Compared with the normal group,there was no significant difference in the expression of HES1 m RNA in the 13 th,15th and 17 th groups?P> 0.05?,The expression of HES1 protein in each group was significantly different from that in normal group?P <0.01?.There was no significant difference in HES1 m RNA between the 16 th group and the untreated group?P> 0.05?,and there was significant difference between the two groups?P <0.01?.The expression of HES1 m RNA was up-regulated in the 16 th and 18 th groups?P <0.01?.12d: The expression of HES1 m RNA and protein in the normal group was significantly different?P <0.01?.The expression of JAG1 m RNA and protein wassignificantly different between the two groups?P <0.01?.The expression of JAG1 m RNA and protein was up-regulated in the 7th,11 th and 12 th groups?P <0.01?.There was no significant difference in HES1 m RNA expression between 9d and 12 d in normal group?P> 0.05?.The expression of HES1 m RNA in the 16 th and 18 th groups was significantly higher than that in the control group?P <0.01?.There was no significant difference in HES1 protein activity between the normal group and the 16 th group and the 18 th group?P> 0.05?.The expression of HES1 protein in the 12 h group was significantly higher than that in the control group?P <0.01?.4.Effects of Bushen Yiqi Huoxue Recipe on Expression of JAG1 m RNA and Protein in 9 and 12 d of Cells in Different Groups Compared with the normal group,the expression of JAG1 m RNA in the 17 th group was not significantly different?P> 0.05?,The expression of JAG1 protein in each group was significantly different from that in normal group?P <0.01?.The expression of JAG m RNA and protein was significantly different between the two groups?P <0.01?.The expression of JAG1 m RNA was up-regulated in the 17 th and 18 th groups?P <0.01?,and the expression of JAG1 protein in the 17 th group was the best?P <0.01?.Compared with the normal group,the expression of JAG1 m RNA and protein was significantly different?P <0.01?.The expression of JAG1 m RNA and protein was significantly different between the two groups?P <0.01?.The expression of JAG1 m RNA and protein was up-regulated in the 7th,11 th and 12 th groups?P <0.01?.There was no significant difference in the expression of JAG1 m RNA between the two groups?P> 0.05?.The expression of HES1 m RNA in the 7,11,12,17 and 18 groups was significantly higher than that in the control group?P <0.01?.The expression of JAG1 protein in the normal group and the transformant group was significantly higher than that in the control group?P <0.01?.The expression of JAG1 protein in 7,11,12,17,18 group was significantly higher than that in the controlgroup?P <0.01?.5.Effects of Bushen Yiqi Huoxue Recipe on the Expression of Notch1 m RNA and Protein in 9 and 12 d of Cells in Different Groups Compared with the normal group,there was no significant difference in Notch1 m RNA between the 1st,4th,5th,6th,7th,8th and 9th groups?P> 0.05?,but the expression of Notch1 protein was significantly different from that of the normal group <0.01);Compared with the untreated group,the expression of Notch1 m RNA and protein was significantly different in each group?P <0.01?.The expression of Notch1 m RNA was up-regulated in the 16 th,17th and 18 th groups?P <0.01?.The expression of JAG1 protein in the 11 th group was the best?P <0.01?Compared with the normal group,the expression of Notch1 m RNA and protein in the 12 d groups were significantly different?P <0.01?.Compared with the untreated group,the expression of Notch1 m RNA and protein was significantly different in each group?P <0.01?.The expression of Notch1 m RNA was up-regulated in the 15 th day?P <0.01?,and the expression of Notch1 protein was up-regulated in the first and second groups?P <0.01?.There was no significant difference in the expression of Notch1 m RNA between the 9th day and the 12 th day?P <0.01?.There was no significant difference in the expression of Notch1 m RNA between the 9th day and the 12 th day?P> 0.05?.The expression of Notch1 protein in normal group and transforming group was significantly lower than that in 9 days?P <0.01?.The expression of Notch1 protein in 9 and 11 groups was significantly higher than that in 9 days?P <0.01?6.The Best Compatibility of Effective Components of Bushen Yiqi Huoxue Decoction The results showed that the best compatibility was A3B1C1D1E1,that is,epimedium(10-6mol / L high dose)antler(10-8mol / L low dose)astragaloside(10-8mol / L low dose)hydroxy safflower yellow A(10-8mol / L low dose)Rehmannide A(10-8mol / L low dose).Conclusion 1.Chinese medicine osteoporosis is "bone atrophy","bone dry" and other areas,the pathogenesis and kidney essence deficiency,spleen and stomach loss,liver qi deficiency and blood stasis network four related,The Treatment often used kidney and spleen,liver and blood,blood circulation and other treatment.2.Bushen Yiqi Huoxue Decoction can improve the bone mineral density of osteoporosis model rats,upregulate the bone formation index,reduce the bone resorption index,confirmed the use of Bushenyiqihuoxue Chinese medicine compound prevention and treatment of osteoporosis have a good effect.3.Bushen Yiqi Huoxue Decoction can increase the expression of RANKL by improving the expression of OPG in osteoporosis rats and play a role in the prevention and treatment of postmenopausal osteoporosis.4.Effect of Bushen Yiqi Huoxue Decoction on the expression of HES1,JAG1 and Notch1 m RNA and protein in bone and renal tissue of osteoporosis rats to prevent postmenopausal osteoporosis.5.(10-8mol / L low dose)Astragaloside(10-8mol / L low dose)Hydroxyl red(10-8mol / L low dose)Antler(10-8mol / L low dose)Astragaloside Flower yellow pigment A(10-8 mol / L low dose)Rehmannide A(10-8 mol / L low dose).6.(The mechanism of BMSCs proliferation and osteogenesis differentiation of BMSCs were related to the up-regulation of HES1,JAG1 and Notch1 m RNA and protein activity in Notch signaling pathway. |
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