| Purpose:To explore the relationship between osteoporosis and the spleen and kidney,based on the theoretical basis of TCM spleen and kidney related "kidney governs bones,generates marrow" and "spleen governs muscles,limbs",and explores the SDF-1/CXCR4 signaling pathway through in vitro experiments The effects of traditional Chinese medicine for nourishing the kidney and strengthening the spleen on the differentiation of BMSCs and MDSCs in rats were observed.In this way,the prevention and treatment effects of traditional Chinese medicines for invigorating the kidney and spleen on osteoporosis are studied and the advantages of traditional Chinese medicine treatment can be exerted to provide a new theoretical basis for clinical prevention and treatment of osteoporosis.Material and method:Sixty SPF female SD rats weighing 200±20g(2 months old)were divided into 6 groups with 10 rats in each group by random number table method which were:normal group,induction fluid group,kidney tonifying Chinese medicine 1+YDY group,kidney tonifying Chinese medicine 2+YDY group,spleen tonifying Chinese medicine +YDY group,kidney tonifying Chinese medicine+YDY group.After 7 days of adaptive feeding,the rats were intraga-stric with the corresponding Traditional Chin ese medicine.The n ormal group and the induced group were given normal saline once at 8 am every day.After 7 days of continuous administration,the corresponding drug-containing serum was prepared.At the end of intragastric administration,blood samples were collected,and drug-containing serum of rats in each group was reserved.BMSCs and MDSCs were divided into the following 6 groups:normal group,induction fluid group,Kidney-tonifying Chinese medicine 1+YDY group,kidney-tonifying Chinese medicine 2+YDY group,spleen-tonifying Chinese medicine+YDY group,kidney-tonifying Chinese medicine +YDY group,and kidney-tonifying Chinese medicine+YDY group.The retained rat serum was filtered,and then BMSCs and MDSCs were cultured in vitro in the corresponding culture medium of rat serum of each group.In the two groups,the normal group was cultured normally,induction solution was added to the induction solution group,and the corresponding drug-containing serum and induction solution were added to other Chinese medicine groups.MTT assay was used to determine the proliferation and differentiation of BMSCs and MDSCs at 24,48 and 72h.BMSCs were cultured for 6d,12d and 18d,respectively.Alizarine red staining was used to observe the formation of mineralized nodules on day 18 of BMSCs culture The protein expressions of BMP-2,SDF-1,CXCR4 and PI3K in SDF1/CXCR4 signaling pathway in each group were detected by ELISA,and the mRNA expressions of SDF-1,CXCR4 and PI3K were detected by qRT-PCR.MDSCs were cultured for 3d,6d,and 12d,respectively.The Desmin expression were observed by immunocytostaining on the 12th day of MDSCs culture.The expression of SDF-1,CKCR4,PI3K,and myosin in the SDF1/CXCR4 signaling pathway of each group of cells was detected by ELISA.For protein expression,the mRNA expression of SDF-1,CXCR4 and PI3K was detected by qRT-PCR.Results:Part I:Based on SDF-1/CXCR4 pathway,the mechanism of regulating rat BMSCs to osteogenic differentiation by Kidney-tonifying and spleen-tonifying TCM was discussedExperiment 1:The effects of drug serum on the proliferation and osteogenic differentiation of rat BMSCs1.Comparison of 24h BMSCs proliferation levels in each group:Compared with the normal group,the proliferation levels of the induction group,the kidney-tonifying Chinese medicine 1+YDY group,and the kidney-tonifying Chinese medicine 2+YDY group were significantly decreased(P<0.01),and the proliferation level of the spleen-tonifying Chinese medicine+YDY group was significantly lower(P<0.01).Compared with the induction group,the proliferation level of the spleen-invigorating Chinese medicine+YDY group was significantly increased(P<0.05),the proliferation level of TCM+YDY group was significantly increased(P<0.01),and the other groups were slightly increased,but not significant(P>0.05).Comparison of the proliferation levels of BMSCs in each group at 48 h:Compared with the normal group,the proliferation levels of each group were significantly decreased(P<0.01);The proliferation level of the Chinese medicine 2+YDY group and the kidney-invigorating and spleen-invigorating Chinese medicine+YDY group decreased,and the proliferation level of the spleen-invigorating Chinese medicine+YDY group was slightly increased,but there was no significant difference(P>0.05).Comparison of the proliferation levels of BMSCs in each group at 72 h:Compared with the normal group,the proliferation levels of the other groups except the kidney-tonifying Chinese medicine 2+YDY group were significantly decreased(P<0.01);The proliferation level in the Chinese medicine 2+YDY group was significantly increased(P<0.01),and the proliferation level in the spleen-invigorating Chinese medicine+YDY group and the kidney-invigorating and spleen-invigorating Chinese medicine+YDY group increased,but it was not significant(P>0.05).The normal group,the induction group,the kidney-tonifying Chinese medicine 1+YDY group,the kidney-tonifying Chinese medicine 2+YDY group,the spleen-invigorating Chinese medicine+YDY group,and the kidney-tonifying and spleen-tonifying Chinese medicine+YDY group all reached the peak at 72h(24h vs 72h P<0.01,48h vs 72h P<0.01).2.The detection results of BMP-2 protein concentration in BMSCs in each experimental group are as follows:Compared with the results of 6d,12d,and 18d,the best detection result was on the 18th day.Results:Compared with the normal group,the BMP-2 content in the kidney-tonifying Chinese medicine 2+YDY group and the kidney-tonifying and spleen-tonifying Chinese medicine+YDY group were significantly increased(P<0.01).,the content of BMP-2 in the spleen-invigorating Chinese medicine+YDY group was significantly decreased(P<0.01),and the BMP-2 content in the induction group and the kidney-tonifying Chinese medicine 1+YDY group was increased,but not significant(P>0.05);The content of BMP-2 in TCM+YDY group was significantly increased(P<0.01),the content of BMP-2 in TCM+YDY group was significantly decreased(P<0.01),the content of BMP-2 in TCM1+YDY group and TCM2+YDY group increased,but not significant(P>0.05).3.Osteogenesis and mineralization of BMSCs in each experimental group after alizarin red staining on the 18th day of culture:After BMSCs were differentiated by osteogenic induction for 18 days,the number of mineralized nodules in the Chinese medicine for nourishing the kidney and spleen+YDY group was the largest,and the volume was also larger than other groups.The group was larger;the mineralized nodules in the induction group,the kidney-tonifying Chinese medicine 1+YDY group,the kidney-tonifying Chinese medicine 2+YDY group,and the spleen-invigorating Chinese medicine+YDY group were more than those in the normal group,and their volume increased significantly;the mineralized nodules in the induction liquid group The number of nodules was less than that in the kidney-tonifying Chinese medicine 1+YDY group and the kidney-tonifying Chinese medicine 2+YDY group,and the volume was smaller and the mineralization level decreased;reduction level.Experiment 2:Effects of tonifying kidney and spleen Chinese medicine-containing serum on osteogenic differentiation of rat BMSCs through SDF-1/CXCR4 signaling pathway1.The detection results of SDF-1 protein concentration in BMSCs in each experimental group:compared with the normal group,the content of SDF-1 in the induction group was significantly increased(P<0.05),and the content of SDF-1 in the other groups was significantly increased(P<0.05).Compared with the induction group,the content of SDF-1 in the kidney-tonifying Chinese medicine 1+YDY group,the kidney-tonifying Chinese medicine 2+YDY group,and the kidney-tonifying and spleen-tonifying Chinese medicine+YDY group had the highest increase(P<0.01).Both of them were significantly increased(P<0.01),and the increa se was the highest in the traditional Chinese medicine for strengthening the kidney and spleen+YDY group(P<0.01).2.The detection results of CXCR4 protein concentration in BMSCs in each experimental group:compared with the normal group,the CXCR4 content in each group was significantly increased(P<0.01):and the increase in the kidney-tonifying and spleen-invigorating Chinese medicine+YDY group was the highest(P<0.01);Compared with the induction group,the content of CXCR4 in the kidney-tonifying Chinese medicine 1+YDY group wa.s significantly increased(P<0.05),and the CXCR4 content in the kidney-tonifying Chinese medicine 2+YDY group and the kidney-tonifying and spleen-invigorating Chinese medicine+YDY group were significantly increased(P<0.01).The content of CXCR4 in the spleen Chinese medicine+YDY group also increased,but it was not significant(P>0.05).3.The detection results of PI3K protein concentration in BMSCs in each experim ental group:compared with the normal group,the content of PI3K in the kidney-tonifying Chinese medicine 1+YDY group,the kidney-tonifying Chinese medicine 2+YDY group,and the kidney-tonifying and spleen Chinese medicine+YDY group were significantly increased(P<0.01),and the highest increase in the kidney-invigorating and spleen-invigorating Chinese medicine+YDY group(P<0.01),the PI3K content in the induction group and the spleen-invigorating Chinese medicine+YDY group also increased,but not significant(P>0.05);compared with the induction group The content of PI3K in the kidney-invigorating Chinese medicine 2+YDY group and the kidney-invigorating and spleen-invigorating Chinese medicine+YDY group were significantly increased(P<0.01),and the PI3K content in the kidney-invigorating Chinese medicine 1+YDY group and the spleen-invigorating Chinese medicine+YDY group also increased,but there was no significant difference.sex(P>0.055).4.The detection results of SDF-1 mRNA in BMSCs in each experimental group:compared with the normal group,the relative expression of SDF-1 in each group was significantly increased(P<0.01),and the expression of the Chinese medicine for nourishing the kidney and strengthening the spleen +YDY group was the best(P<0.01);Compared with the induction group,the relative expression of SDF-1 in the kidney-tonifying Chinese medicine 1+YDY group,the kidney-tonifying Chinese medicine 2+YDY group,and the kidney-tonifying and spleen-tonifying Chinese medicine-YDY group were significantly increased(P<0.01)3 The relative expression of SDF-1 in spleen Chin ese medicine+YDY group increased,but it wasnot significant(P>0.05).5.The detection results of CXCR4 mRNA in BMSCs in each experimental group:compared with the normal group,the relative expression of CXCR4 in each group was significantly increased(P<0.01),and the best expression was in the Chinese medicine for tonifying the kidney and spleen+YDY group(P<0.01);Compared with the induction group,the relative expression of CXCR4 in the kidney-tonifying Chinese medicine 1+YDY group and the kidney-tonifying spleen Chinese medicine+YDY group was significantly increased(P<0.01):and the CXCR4 in the kidney-tonifying Chinese medicine 2+YDY group and the spleen-invigorating Chinese medicine+YDY group was significantly increased(P<0.01).Therelative expression level increased,but not significant(P>0.05).6.The detection results of PI3K mRNA in BMSCs in each experimental group:compared with the mormal group,the relative expression of PI3K in each group was significantly increased(P<0.01),and the best expression was in the Chinese medicine for tonifying the kidney and spleen+YDY group(P<0.01);Compared with the induction group,the relative expression of PI3K in the kidney-tonifying Chinese medicine 1+YDY group,the kidney-tonifying Chinese medicine 2+YDY group,and the kidney-tonifying and spleen-tonifying Chinese medicine+YDY group were significantly increased(P<0.01)The relative expression level was also signficantly increased(P<0.05).Part Ⅱ:Based on the SDF-1/CXCR4 pathway to explore the mechanism of traditional Chinese medicine for nourishing the kidney and strengthening the spleen to regulate the differentiation of rat MDSCs into myoblastsExperiment 3:Effects of drug-containing serum in each group on the proliferation and myogenic differentation of rat MDSCs1.Comparison of 24h MDSCs proliferation levels in each group:Compared with the normal group,the proliferation level of the induction group and the spleen-invigorating Chinese medicine+YDY group was slightly decreased,and the proliferation level of the kidney-tonifying Chinese medicine 2+YDY group was slightly increased,but neither had a significant effect.Compared with the induction group,the proliferation level of the kidney-tonifying Chinese medicine 2+YDY group and the kidney-tonifying and spleen-tonifying Chinese medicine+YDY group were significantly increased(P>0.05).High(P<0.01),the proliferation level of kidney-tonifying Chinese medicine 1+YDY group also increased,but it was not significant(P>0.05).Comparison of the proliferation levels of MDSCs in each group at 48 hours:Compared with the normal group,the proliferation levels of the kidney-tonifying Chinese medicine 2+YDY group and the kidney-tonifying and spleen-tonifying Chinese medicine+YDY group were significantly increased(P<0.01).There was no significance(P>0.05);compared with the induction group,the proliferation level of the traditional Chinese medicine for nourishing the kidney and spleen+YDY group was significantly increased(P<0.01),and the group of traditional Chinese medicine for nourishing the kidney 2+YDY increased,but it was not significant(P>0.05).Comparison of the proli feration levels of MDSCs in each group at 72 h:Compared with the normal group,the proliferation levels of the kidney-tonifying Chinese medicine 1+YDY group and the kidney-tonifying and spleen-tonifying Chinese medicine+YDY group were significantly increased(P<0.01),and the proliferation level of the kidney-tonifying Chinese medicine 2+YDY group was significantly higher.Compared with the induction group,the proliferation,level of the kidney-tonifying Chinese medicine 1+YDY group and the kidney-tonifying spleen Chinese medicine+YDY group were significantly increa sed(P<0.01):and the proliferation level of the kidney-tonifying Chinese medicine 2+YDY group was significantly increased(P<0.05).The normal group,the induction group,the kidney-tonifying Chinese medicine 1+YDY group:the kidney-tonifying Chinese medicine 2+YDY group,the spleen-invigorating Chinese m edi cine+YDY group,and the kidney-tonifying and spleen-tonifying Chinese medicine+YDY group all reached the peak at 72h(24h vs 48h P<0.05,24h vs 72h).P<0.01).2.The detection results of myosin concentration in MDSCs in each experimental group:compared with 3d,6d,and 12d,the detection result on the 12th day was the best,and the 12d result:compared with the normal group,the myosin content in each group was significantly increa sed(P<0.01),and the myosin content in the kidney-invigorating and spleen-invigorating Chinese medicine+YDY group increased the best(P<0.01);The content of myosin was signficantly increased(P<0.01),and the myosin content was also increased in the kidney-tonifying Chinese medicine 1+YDY group and the spleen-invigorating Chinese medicine+YDY group,but there was no significant difference(P>0.05).3.The results of immunocytochemical staining after the 12th day of culture of MDSCs in each experim ental group:Compared with the normal group,the mean fluorescence density of each group was significantly enhanced(P<0.05),and the other groups were significantly enhanced(P<0.01).Compared with the induction group,the kidney-tonifying Chinese medicine 2+YDY group was significantly enhanced(P<0.05),the traditional Chinese medicine for nourishing the kidney and spleen+YDY group was significantly enhanced(P<0.01),and the other groups were enhanced:but there was no significant difference(P>0.05).Experiment 4:Effects of tonifying kidney and spleen Chinese medicine-containing serum on myogeoic differention of rat MDSCs through SDF-1/CXCR4 signalingPathway1.The detection results of SDF-1 protein concentration in MDSCs in each experimental group:Compared with the normal group,the content of SDF-1 in the kidney-tonifying Chinese medicine 1+YDY group,the kidney-tonifying Chinese medicine 2+YDY group,and the kidney-tonifying and spleen-tonifying Chinese medicine+YDY group were significantly increased.High(P<0.01):the content of SDF-1 in the induction group and the spleen-invi gorating Chinese medicine+YDY group was also significantly increased(P<0.05),and the SDF-1 content in the kidney-invigorating and spleen-invi gorating Chinese medicine+YDY group increased most significantly(P<0.01);Compared with the induction group,the content of SDF-1 in TCM 1+YDY group,TCM 2+YDY group,TCM+YDY group were significantly increased(P<0.01):and SDF-1 in TCM+YDY group was significantly increas ed(P<0.01).1 content also increased sli ghtly,but there was no significant difference(P>0.05).2.The detection results of CXCR4 protein concentration in MDSCs in each experimental group:compared with the normal group,the content of CXCR4 in the other groups except the spleen-invigorating Chinese medicine+YDY was significantly increas ed(P<0.01),and the spleen-invigorating Chinese medicine+YDY was significantly increased(P<0.01).Compared with the induction group,the content of CXCR4 in the kidney-tonifying Chinese medicine 1+YDY group and the kidney-tonifying and spleen Chinese medicine+YDY group were significantly increased(P<0.01):and the kidney-tonifying Chinese medicine 2+YDY The content of CXCR4 in the group also increased slightly,but there was no significant difference(P>0.05).3 The results of the detection of PI3K protein concentration in MDSCs in each experimental group:Compared with the normal group,the PI3K content in the other groups except the induction group was significantly increased(P<0.01):and the PI3K content in the kidney-invigorating and spleen-invigorating Chinese medicine+ YDY group Compared with the induction group,the content of PI3K in TCM 1+YDY group and TCM+YDY group were significantly increased(P<0.01),and the content of PI3K in TCM+YDY group was also significantly increased(P<0.01).significantly increased(P<0.05),and the content of PI3K in the kidney-t oni fying Chinese medicine 2+YDY group also increased,but there was no significant difference(P>0.05).4.The detection results of SDF-1 mRNA in MDSCs in each experimental group:compared with the normal group,the relative expression of SDF-1 in each group was significantly increased(P<0.01),and the expression of the Chinese medicine for nourishing the kidney and spleen+YDY group was the best(P<0.01);Compared with the induction group,the relative expression of SDF-1 in the kidney-tonifying Chinese medicine 1+YDY group and the kidney-tonifying and pleen-tonifying Chinese medicine+YDY group were significantly increased(P<0.01),and the SDF-1 in the kidney-tonifying Chinese medicine 2+YDY group was significantly increased(P<0.01).1 The relative expression level was significantly increased(P<0.05),and the relative expression level of SDF-1 in the spleen-strengthening Chinese medicine+YDY group was increased,but there was no significant difference(P>0.05).5.The detection results of CXCR4 mRNA in MDSCs in each experimental group:compared with the normal group,the relative expression of CXCR4 in each group was significantly increased(P<0.01),and the best expression was in the Chinese medicine for nourishing the kidney and spleen+YDY group(P<0.01));compared with the induction group,the relative expression of CXCR4 in the kidney-tonifying Chinese medicine 1+YDY group and the kidney-tonifying spleen Chinese medicine+YDY group were significantly increased(P<0.01),and the relative expression of CXCR4 in the kidney-tonifying Chinese medicine 2+YDY group was significantly increased(P<0.05),the relative expression of CXCR4 in the spleen-strengthening Chinese medicine+YDY group increased,but there was no significant difference(P>0.05).6.The detection results of PI3K mRNA in MDSCs in each experimental group:compared with the normal group,the relative expression of PI3K in each group was significantly increased(P<0.01),and the expression of the traditional Chinese medicine for invigorating the kidney and spleen+YDY group was the best(P<0.01);Compared with the induction group,the relative expression of PI3K in the kidney-tonifying Chinese medicine 1+YDY group,the kidney-tonifying Chinese medicine 2+YDY group,and the kidney-tonifying and spleen-tonifying Chinese medicine+YDY group were significantly increased(P<0.01).The relative expression level increased,but there was no significant difference(P>0.05).Conclusion:1.BMSCs of normal rats can express BMP-2,SDF-1,CXCR4 and PI3K related factors during differentiation;Normal rat MDSCs can express myosin,SDF-1,CXCR4 and PI3K related factors during differentiation.2.The serum containing traditional Chinese medicine for tonifying kidney and strengthening spleen can promote the proliferation and osteogenic dffferentiation of BMSCs,as well as the proliferation and myoblast differentiation of MDSCs,and its mechanism is related to the regulation of sdF-1,CXCR4 and PI3K protein and mRNA expressions in SDF-1/CXCR4 signaling pathway.3.Kidney tonfying and spleen tonifying serum could improve protein content and mRNA expression of BMP-2,SDF-1,CXCR4 and PI3K during BMSCs differentiation in rats;The protein contents and mRNA expressions of myosin,SDF-1,CXCR4 and PI3K during the differentiation of MDSCs in rats were increased to achieve the purpose of prevention and treatment of osteoporosis.4.Serum containing traditional Chinese medicines for invigorating kidney and spleen promotes the osteogenic differentiation of BMSCs and the myogenic differentiation of MDSCs.In this experiment,Bushen Jianpi recipe promotes the osteogenic differentiation of BMSCs and the myogenic differentiation of MDSCs is the best.The traditional Chinese medicine group of tonifying the kidney and strengthening the spleen was better than the traditional Chinese medicine group that simply adopted the legislation of "tonifying the kidney" and "strengthening the spleen",which further indicated that "the method of nourishing the kidney and strengthening the spleen" may be more effective in the prevention and treatment of osteoporosis.5.The traditional Chinese medicines used in this experiment can promote the differentiation of BMSCs and MDSCs,which confirms the theory of traditional Chinese medicine that"kidney governs bones and generates marrow;spleen governs muscles and limbs",and further confirms that kidney deficiency is the root cause of osteoporosis.,Spleen deficiency is the key to the pathogenesis of osteoporosis,and the treatment should focus on nourishing the kidney and strengthening the spleen. |
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