| Purpose: Based on the theory of "correlation between spleen and kidney",this paper discusses the mechanism of Bushen Jianpi formula and its disassembled formula promoting the osteogenic differentiation of BMSCs in rats,and reveals its mechanism of interfering with disuse osteoporosis by promoting bone formation.Material and method:Thirty-eight SPF rats,male,6 weeks old,body mass(220±10)g,were randomly divided into four groups: normal group,kidney tonic group,spleen tonic group and kidney and spleen tonic group,of which 14 rats were in the normal group(8 rats were prepared with drug-containing serum and 6 rats were prepared with bone marrow mesenchymal stem cells),and the remaining 8 rats were in each group.Based on the conversion ratio of equivalent dose between human and animal body surface area ratios,rats in the kidney tonic group,spleen tonic group and kidney and spleen tonic group were gavaged with the equivalent doses of drugs administered to the population,3.24 g/kg in the kidney tonic group,2.43 g/kg in the spleen tonic group and 5.67 g/kg in the kidney and spleen tonic group,while rats in the normal group were given the same The rats in the normal group were given the same volume of distilled water(the 6 rats used for the preparation of bone marrow MSCs were not gavaged),and 2 ml of the drug was administered once a day to the rats requiring gavage for 7 d.2 h after gavage on the 7th day,the rats were anesthetized,the abdomen of the rats was cut open after sterilization with surgical scissors,and blood was collected from the abdominal aorta,and the centrifuge tubes with blood were stored at room temperature and left for 2 h.The centrifuge was set at 3000 rpm for 20 min.The supernatant portion of the tube was aspirated,filtered,de-bacterized,inactivated and divided using a needle filter,and finally stored in a refrigerator at-80℃.The remaining 6 rats in the normal group were put to death by cervical dislocation method and soaked in 75% alcohol for 10 min,gauze dipped in iodophor was wiped on the rats’ body for 2 times for comprehensive disinfection to ensure aseptic conditions for bone extraction,the rats were placed in the ultra-clean table,their femur and tibia were removed,and the muscle fascia tissue attached to the bone was shaved,and LG-DMEM medium containing 10% fetal bovine serum was aspirated into the bone using a syringe.-DMEM culture solution containing 10% fetal bovine serum was injected into the bone marrow cavity,and the rinsing was repeatedly performed until the color changed to white and the rinsing was stopped to ensure complete rinsing.Bone marrow mesenchymal stem cells were cultured in primary culture to the third generation.The proliferation of each group of osteoblasts was detected by tetramethylazole salt colorimetric method(MTT method),and the optimal concentration of each drug-containing serum for the intervention of bone marrow MSCs was screened.The results were analyzed and divided into blank serum group(blank serum),induction group(osteogenic induction solution),spleen-containing serum group(spleen-containing serum plus osteogenic induction solution),kidney-containing serum group(kidney-containing serum plus osteogenic induction solution),and kidney-containing serum group(kidney-containing serum plus osteogenic induction solution)for 14 days.Alkaline phosphatase(ALP)activity in the supernatant of rat bone marrow MSCs after 3,7 and 14 days of osteogenic induction,and the formation of mineralized nodules in each group of rat bone marrow MSCs was detected by alizarin red staining.-Smad5,BMP-2 and Osterix protein expression in rat bone marrow MSCs.Results:1.Effects of different concentrations of Kidney and Spleen Formula and its split formula containing serum on the proliferation activity of BMSCs1.1 Effect of different concentrations of Jianshuifang containing serum on the proliferative activity of BMSCs at different time points At 24 hours of incubation,the cell proliferation activity was significantly higher in the 5%spleen-containing serum group compared with the 5% blank control serum group(P < 0.01);the cell proliferation activity was significantly higher in the 10% spleen-containing serum group compared with the 10% blank control serum group(P < 0.01);and the cell proliferation activity was higher in the 20% spleen-containing serum group compared with the 20% blank control serum group(P < 0.05).At 48 hours of incubation,the cell proliferation activity was significantly higher in the 5%spleen-containing serum group compared with the 5% blank control serum group(P < 0.01);the cell proliferation activity was significantly higher in the 10% spleen-containing serum group compared with the 10% blank control serum group(P < 0.01);and the cell proliferation activity was significantly higher in the 20% spleen-containing serum group compared with the20% blank control serum group(P < 0.01).At 72 hours of incubation,the cell proliferation activity was significantly higher in the 5%spleen-containing serum group compared with the 5% blank control serum group(P < 0.01);compared with the 10% blank control serum group,the cell proliferation activity was higher in the 10% spleen-containing serum group,but there was no significant difference;compared with the 20% blank control serum group,the cell proliferation activity was higher in the 20%spleen-containing serum group,but there was no significant difference.1.2 Effect of different concentrations of Kidney Tonic Formula-containing serum on the proliferative activity of BMSCs at different time points At 24 hours of incubation,there was no significant difference in cell proliferation activity between the 5% kidney tonic-containing serum group compared with the 5% blank control serum group;compared with the 10% blank control serum group,there was no significant difference in cell proliferation activity between the 10% kidney tonic-containing serum group;compared with the 20% blank control serum group,there was no significant difference in cell proliferation activity between the 20% kidney tonic-containing serum group.At 48 hours of incubation,the cell proliferation activity was increased in the 5% kidney supplement-containing serum group compared with the 5% blank control serum group(P <0.05);the cell proliferation activity was significantly increased in the 10% kidney supplement-containing serum group compared with the 10% blank control serum group(P <0.01);the cell proliferation activity was significantly increased in the 20% kidney supplement-containing serum group compared with the 20% blank control serum group(P <0.01).At 72 hours of incubation,there was no significant difference in the cell proliferation activity of the 5% kidney tonic-containing serum group compared with the 5% blank control serum group;the cell proliferation activity of the 10% kidney tonic-containing serum group was increased compared with the 10% blank control serum group(P < 0.05);the cell proliferation activity of the 20% kidney tonic-containing serum group was not significantly different compared with the 20% blank control serum group.1.3 Effect of different concentrations of Kidney Tonic and Spleen Formula-containing serum on the proliferative activity of BMSCs at different time points At 24 hours of incubation,the cell proliferation activity was significantly higher in the 5%kidney tonic and spleen-containing serum group compared with the 5% blank control serum group(P < 0.01);the cell proliferation activity was significantly higher in the 10% kidney tonic and spleen-containing serum group compared with the 10% blank control serum group(P < 0.01);the cell proliferation activity was significantly higher in the 20% kidney tonic and spleen-containing serum group compared with the 20% blank control serum group(P < 0.01);the cell proliferation activity was significantly higher in the 20% kidney tonic and spleen-containing serum group(P < 0.01).At 48 hours of incubation,the cell proliferation activity was significantly higher in the 5%kidney and spleen supplementation serum group compared with the 5% blank control serum group(P < 0.01);the cell proliferation activity was significantly higher in the 10% kidney and spleen supplementation serum group compared with the 10% blank control serum group(P <0.01);the cell proliferation activity was not significantly different in the 20% kidney and spleen supplementation serum group compared with the 20% blank control serum group.There was no significant difference in cell proliferation activity between the 20% kidney and spleen tonic serum and the 20% blank control serum group.At 72 hours of incubation,the cell proliferation activity was significantly higher in the 5%kidney and spleen tonic-containing serum group compared with the 5% blank control serum group(P < 0.01);compared with the 10% blank control serum group,there was no significant difference in the cell proliferation activity in the 10% kidney and spleen tonic-containing serum group;compared with the 20% blank control serum group,there was no significant difference in the cell proliferation activity in the 20% kidney and spleen tonic-containing serum group.2.The effect of osteogenesis induction on the ALP content of cell supernatant at different time points of tonics and spleen formula and its dismantled serum Compared with the blank serum group,the cell supernatant ALP content was significantly higher in the induction group at 3,7 and 14 days of osteogenesis induction(P < 0.01);compared with the induction group,the cell supernatant ALP content was significantly higher in the tonic kidney-containing serum and tonic kidney and spleen-containing serum groups at3 days of osteogenesis induction(P <0.01),and the cell supernatant ALP content was higher in the spleen-containing serum groups at 7 and 14 days of osteogenesis induction(P<0.01).(P < 0.05),and the supernatant ALP levels were significantly higher in the osteogenic induction 7-day and 14-day kidney tonic-containing serum groups and the kidney and spleen tonic-containing serum groups(P < 0.01),and the supernatant ALP levels were significantly higher in the osteogenic induction 7-day kidney tonic-containing serum group compared with the spleen tonic-containing serum group(P < 0.01),and the supernatant ALP levels were higher in the osteogenic induction 14-day kidney tonic-containing serum group(P < 0.01).The supernatant ALP content was increased(P<0.05);compared with the kidney and spleen tonic-containing serum group,the supernatant ALP content of cells in the spleen-containing serum group was significantly decreased(P < 0.01)in the 3,7 and 14 days of osteogenesis induction,and the supernatant ALP content of cells in the 7 and 14 days of osteogenesis induction tonic-containing serum group was decreased(P<0.05).3.Effect of kidney tonic and spleen formula and its split formula containing serum on osteogenesis-induced mineralized nodule formation in BMSCs with alizarin red staining BMSCs osteogenesis was induced for 14 days,and alizarin red staining was performed,which could be observed microscopically.Compared with the blank serum group,mineralized nodule formation was seen in the induced group;compared with the kidney tonic-containing serum group and the spleen-containing serum group,mineralized nodule formation was more obvious in the kidney tonic and spleen-containing serum group.4.Effect of serum containing Bushen Jianpi Recipe and its disassembled Recipe on the expression of BMP-2 in rat BMSCs Compared with the blank serum group,the expression of BMP-2 in the induction group was significantly higher(P < 0.01);Compared with the induction group,the expression of BMP-2 in the serum group containing invigorating the spleen,the serum group containing invigorating the kidney and the serum group containing invigorating the kidney and spleen was significantly higher(P < 0.01);Compared with Jianpi medicated serum group,the expression of BMP-2 in Bushen medicated serum group was significantly higher(P < 0.01);Compared with the serum containing medicine for tonifying the kidney and spleen,the expression of BMP-2 in the serum containing medicine for tonifying the spleen group and the serum containing medicine for tonifying the kidney group decreased significantly(P < 0.01).5.Effect of serum containing Bushen Jianpi Recipe and its disassembled Recipe on the expression of Smad1 in rat BMSCs Compared with the blank serum group,the expression of Smad1 in the induction group was significantly higher(P < 0.05);Compared with the induction group,the expression of Smad1 in the serum containing invigorating the spleen,the serum containing tonifying the kidney and the serum containing invigorating the kidney and spleen was significantly higher(P < 0.01);Compared with the serum containing medicine for tonifying the kidney and spleen,the expression of Smad1 in the serum containing medicine for tonifying the spleen group and the serum containing medicine for tonifying the kidney group was significantly higher(P < 0.01).6.Effect of serum containing Bushen Jianpi Recipe and its disassembled Recipe on the expression of p-smad1 in rat BMSCs Compared with the blank serum group,the expression of p-smad1 in the induction group was significantly higher(P < 0.01);Compared with the induction group,the expression of p-smad1 in Jianpi medicated serum group,Bushen medicated serum group and Bushen Jianpi medicated serum group was significantly higher(P < 0.01);The expression of p-smad1 in the serum group containing tonifying the kidney was significantly higher than that in the serum group containing tonifying the spleen(P < 0.01);The expression of p-smad1 in the serum containing tonifying kidney and spleen was significantly lower than that in the serum containing tonifying kidney and spleen(P < 0.01).7.Effect of serum containing Bushen Jianpi Recipe and its disassembled Recipe on the expression of Smad5 in rat BMSCs Compared with the induction group,the expression of Smad5 in the serum containing Jianpi and Bushen Jianpi decreased significantly(P < 0.01);Compared with the serum containing medicine for invigorating the spleen group,the expression of Smad5 in the serum containing medicine for invigorating the kidney and spleen group decreased significantly(P < 0.01).8.Effect of serum containing Bushen Jianpi Recipe and its disassembled Recipe on the expression of p-smad5 in rat BMSCs Compared with the blank serum group,the expression of p-smad5 in the induction group was significantly higher(P < 0.01);Compared with the induction group,the expression of p-smad5 in the serum group containing invigorating the spleen,the serum group containing invigorating the kidney and the serum group containing invigorating the kidney and spleen was significantly higher(P < 0.01);Compared with the serum containing medicine in the spleen strengthening group,the expression of p-smad5 in the serum containing medicine for tonifying the kidney group was significantly higher(P < 0.01);The expression of p-smad5 in the serum containing tonifying kidney and spleen was significantly lower than that in the serum containing tonifying kidney and spleen(P < 0.01).9.Effect of serum containing Bushen Jianpi Recipe and its disassembled Recipe on osterix expression in rat BMSCs Compared with the blank serum group,the expression of osterix in the induction group was significantly higher(P < 0.01);Compared with the induction group,the expression of osterix in the serum containing invigorating the spleen,the serum containing tonifying the kidney and the serum containing invigorating the kidney and spleen was significantly higher(P < 0.01);The expression of osterix in the serum containing tonifying kidney was significantly higher than that in the serum containing invigorating spleen(P < 0.01);Compared with the serum containing medicine for tonifying the kidney and spleen,the expression of osterix in the serum containing medicine for tonifying the spleen group and the serum containing medicine for tonifying the kidney group decreased significantly(P < 0.01).Conclusion.1.Bushen Jianpi Recipe,Jianpi Recipe and Bushen recipe can promote the osteogenic differentiation of BMSCs in rats,and the effect of Bushen Jianpi Recipe is the best.2.Possible mechanism of Bushen Jianpi Recipe promoting osteogenic differentiation of rat BMSCs: regulate BMP-2 to activate Smad 1 / p-smad 1 / Smad 5 / p-smad 5 / osterix signal pathway,regulate the expression of osteoblast protein,promote bone formation and induce osteogenic differentiation.The mechanism of Jianpi Recipe and Bushen recipe is the same as Bushen Jianpi Recipe. |
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