| Purpose:Based on the theory of "spleen kidney correlation" in traditional Chinese medicine,to explore the prevention and treatment of osteo-sarcopenia.Using the different treatment methods of tonifying the kidney,tonifying the spleen,and tonifying the kidney and the spleen in traditional Chinese medicine,in vitro experiments were conducted to investigate the effects of the SDF-1/CXCR4 signaling pathway on the myogenic differentiation of rat BMSCs.Cell morphology and the expression of SDF-1,CXCR4,JAK2,ROS,COX-2,myosin related factor proteins,and SDF-1,CXCR4,JAK2 m RNA in the SDF-1/CXCR4 signaling pathway were observed to investigate the preventive and therapeutic effects of different treatment methods on osteo-sarcopenia.Utilize the advantages of traditional Chinese medicine treatment to provide new theoretical basis for the clinical prevention and treatment of osteo-sarcopenia.Material and method:Sixty SPF grade SD female rats weighing 200 ± 20g(2months old)were randomly divided into 6 groups using random digital table method,with 10 rats in each group.They were divided into: normal group,myogenic induction group,kidney tonifying group 1,kidney tonifying group 2,spleen tonifying group,and kidney and spleen tonifying group.After adaptive feeding of rats for 7days,the rats were gavaged with corresponding traditional Chinese medicine.The normal group and induced group were both treated with ultrapure water and administered once at 8 am every day.After continuous administration for 7 days,corresponding drug-containing serum was prepared.After gavage,blood samples were collected and the drug-containing serum of each group of rats was retained.Divide the purchased rat BMSCs into the following 6 groups: normal group,myogenic induction group,kidney tonifying group 1,kidney tonifying group 2,spleen tonifying group,and kidney and spleen tonifying group for cells resuscitation,proliferation,and subculture.Filter the retained rat serum,and then culture BMSCs in vitro using the corresponding rat serum culture medium for each group.The normal group is cultured normally,and the induction group is supplemented with myogenic induction solution.Other traditional Chinese medicine groups are supplemented with corresponding drug-containing serum and induction solution.MTT method was used to measure the proliferation and differentiation of BMSCs at 24,48,and 72 hours.BMSCs were cultured for 6d,12 d,and 15 d respectively.The expression of Desmin was observed by immunocytochrome staining on the 15 th day of BMSCs culture.The protein expression of SDF-1,CXCR4,JAK2,ROS,COX-2 and myosin in the SDF-1/CXCR4 signaling pathway was detected by ELISA in each group of cells.The m RNA expression of SDF-1,CXCR4,and JAK2 was detected by q RT-PCR.Results:Part 1: The effects of traditional Chinese medicine for tonifying the kidney and tonifying the spleen on the proliferation and myogenic differentiation of BMSCs Experiment 1: The effect of serum from drug-containing rats in each group on the proliferation of BMSCs1.Comparison of proliferation levels of 24-hour BMSCs in each groupCompared with the normal group,the proliferation levels of the induction group,kidney tonifying group 1,kidney tonifying group 2,and spleen tonifying group decreased,the proliferation level of kidney and spleen tonifying group slightly increased.There were statistically significant differences between the normal group and the induction group,between the normal group and kidney tonifying group 2(P<0.01).Compared with the induction group,the proliferation levels of the medication groups increased,and there were statistically significant differences between the induction group and kidney tonifying group 1,between the induction group and kidney and spleen tonifying group(P<0.05).2.Comparison of proliferation levels of 48-hour BMSCs in each groupCompared with the normal group,the proliferation levels of the induction group and the medication groups decreased,and the differences were statistically significant(P<0.01).Compared with the induction group,the proliferation level outside kidney tonifying group 1 was higher than the induction group.The proliferation levels in kidney tonifying group 2,spleen tonifying group,and kidney and spleen tonifying group were all lower than that in the induction group,and the difference between kidney and spleen tonifying group and the induction group was statistically significant(P<0.05).3.Comparison of proliferation levels of 72-hour BMSCs in each groupCompared with the normal group,the proliferation levels of the induction group and the medication groups decreased,and the differences were statistically significant(P<0.01).Compared with the induction group,the proliferation levels of kidney tonifying group 1,spleen tonifying group,and kidney and spleen tonifying group all decreased,the proliferation level of kidney tonifying group 2 slightly increased,the differences were not statistically significant(P>0.05).Experiment 2: The effect of drug-containing rat serum in each group on myogenic differentiation of BMSCs1.Myosin test results1.1 Myosin detection results on the 6th dayCompared with the normal group,the expression levels of the induction group and the medication groups were significantly higher than those of the normal group,and the differences were statistically significant(P<0.05).Compared with the induction group,the expression levels of the medication groups were better than that of the induction group.Except for kidney tonifying group 2,the differences were statistically significant(P<0.05).1.2 Myosin detection results on the 12 th dayCompared with the normal group,the expression levels of the induction group and the medication groups were significantly higher than those of the normal group,and the differences were statistically significant(P<0.01).Compared with the induction group,the expression levels of the medication groups were better than that of the induction group,and the differences were statistically significant(P<0.01).1.3 Myosin detection results on the 15 th dayCompared with the normal group,the expression levels of the induction group,kidney tonifying group 1,kidney tonifying group 2,and spleen tonifying group were significantly reduced.Kidney and spleen tonifying group was slightly higher than the normal group.Except for spleen tonifying group,the differences were statistically significant(P<0.05).Compared with the induction group,the expression levels in the medication groups were significantly increased,and the differences were statistically significant(P<0.05).2 The average fluorescence intensity of Desmin expression in cells detected by immunofluorescence assayThe average intensity of cellular immune fluorescence in each group: Compared with the normal group,the induction group and medication groups were significantly higher than the normal group,and the differences were statistically significant(P<0.01).Compared with the induction group,the medication groups were significantly higher than the induction group,and the differences were statistically significant(P<0.01).Part 2: Exploring the mechanism of different traditional Chinese medicine treatments regulating the differentiation of rat BMSCs into muscles based on the SDF-1/CXCR4 pathwayExperiment 3: The effect of medicated serum containing different treatment methods on myogenic differentiation of rat BMSCs through the SDF-1/CXCR4 signaling pathway1 ELISA results1.1 Detection results of SDF-1 concentration in cells supernatant of each groupCompared with the normal group,the induction group,kidney tonifying group 1,kidney tonifying group 2,and kidney and spleen tonifying group were significantly better than the normal group.The results of spleen tonifying group were lower than the normal group.There were statistically significant differences between the normal group and the induction group,between the normal group and kidney and spleen tonifying group(P<0.05).Compared with the induction group,kidney tonifying group1,kidney tonifying group 2,and spleen tonifying group were lower than the induction group,kidney and spleen tonifying group was higher than the induction group.The difference between the induction group and spleen tonifying group was statistically significant(P<0.05).1.2 Detection results of CXCR4 concentration in cells supernatant of each groupCompared with the normal group,the expression levels in the induction group and the medication groups were significantly increased.Except for the induction group and spleen tonifying group,the differences were statistically significant(P<0.05).Compared with the induction group,except for spleen tonifying group which was lower than the induction group,all other medication groups were significantly higher than the induction group,the difference between the induction group and kidney and spleen tonifying group was statistically significant(P<0.01).1.3 Detection results of JAK2 concentration in cells supernatant of each groupCompared with the normal group,the induction group remained basically unchanged,the medication groups were significantly higher than the normal group.Except for the induction group,the differences were statistically significant(P<0.05).Compared with the induction group,the medication groups showed a significant increase,and the differences were statistically significant(P<0.05).1.4 Detection results of ROS concentration in cells of each groupCompared with the normal group,except for spleen tonifying group,all other groups were lower than the normal group,and the differences were statistically significant(P<0.01).Compared with the induction group,the medication groups were higher than the induction group.Except for kidney tonifying group 1,the differences were statistically significant(P<0.01).1.5 Detection results of COX-2 concentration in cells of each groupCompared with the normal group,the induction group and the medication groups showed a significant increase.Except for spleen tonifying group,there were statistically significant differences(P<0.05).Compared with the induction group,except for the slight increase in kidney tonifying group 2,the other medication groups were all lower than the induction group.There were statistically significant differences between the induction group and kidney tonifying group 1,between the induction group and spleen tonifying group(P<0.05).2 qRT-PCR results2.1 Detection of SDF-1 m RNA expression in myoblasts using q RT-PCR methodCompared with the normal group,the induction group and the medication groups showed a significant increase,and the difference between kidney and spleen tonifying group and the normal group was statistically significant(P<0.05).Compared with the induction group,the medication groups showed statistically significant increase(P<0.05).2.2 Detection of CXCR4 m RNA expression in myoblasts using qRT-PCR methodCompared with the normal group,the expression levels in the induction group and the medication groups were significantly increased,and the differences were statistically significant(P<0.05).Compared with the induction group,the medication groups showed an increase.There were statistically significant differences between the induction group and kidney tonifying group 1,and between the induction group and kidney and spleen tonifying group(P<0.05).2.3 Detection of JAK2 m RNA expression in myoblasts using qRT-PCR methodCompared with the normal group,the expression levels in the induction group and the medication groups were significantly increased.There was a statistically significant difference between the normal group and kidney tonifying group 1,and between the normal group and kidney and spleen tonifying group(P<0.05).Compared with the induction group,the expression levels in the medication groups were significantly increased,there was a statistically significant difference between the induction group and kidney and spleen tonifying group(P<0.05).Conclusion:1.During the myogenic differentiation process of normal rat BMSCs,SDF-1,CXCR4,JAK2,ROS,and COX-2 related factors can be expressed.2.Traditional Chinese medicine with different treatment methods can promote the proliferation and myogenic differentiation of BMSCs,and its mechanism of action is related to regulating the expression of SDF-1,CXCR4,JAK2,ROS,COX-2 proteins and SDF-1,CXCR4,and JAK2 m RNA in the SDF-1/CXCR4 signaling pathway.3.The different treatment methods of tonifying the kidney,tonifying the spleen,and tonifying the kidney and the spleen can promote the myogenic differentiation ofBMSCs,it confirms the feasibility of preventing and treating sarcopenia-osteoporosis based on the theory of "spleen kidney correlation" in traditional Chinese medicine and starting from the relationship between innate and acquired in traditional Chinese medicine. |
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