| ObjectiveThis study used ovariectomized(OVX)rats as an osteoporosis(OP)model to investigate the mechanism of the Qianggu Capsule,a traditional Chinese medicine for tonifying the kidneys,in regulating the SDF-1/CXCR4 axis to promote the homing of bone marrow mesenchymal stem cells(BMSCs)and intervene in OP in OVX rats.The study revealed the differential effects of the Qianggu Capsule on the lumbar vertebrae(bone)and mandible(teeth),providing biological evidence for the traditional Chinese medicine theory of "kidneys governing bone and generating marrow" and "teeth as the surplus of bones," and offering scientific support for the use of tonifying kidney herbs in the treatment of OP.MethodsAfter a two-week acclimatization period,80 ten-week-old female SD rats were randomly assigned into four groups using a random number table:a baseline group(8 rats),a blank group(16 rats),a sham surgery group(16 rats),and a model group(40 rats).The baseline group underwent no treatment and was euthanized under anesthesia to collect the fifth lumbar vertebrae and right mandibles,which were fixed in 4%paraformaldehyde for subsequent analysis.The model group underwent ovariectomy surgery to induce kidney-deficient osteoporosis in rats.The sham surgery group underwent the same procedure,except for the removal of some fatty tissue around both ovaries.After the surgery,eight rats died,leaving 32 rats in the model group.Three months after ovariectomy or sham surgery,eight rats were randomly selected from the blank,sham surgery,and model groups and euthanized under anesthesia to collect the fifth lumbar vertebrae and right mandibles.The remaining rats in the model group were randomly divided into three groups of eight rats each:an ovariectomy group(OVX group,6 months),an OVX+estrogen group(positive control group),and a castration+Qianggu Capsule group(Qianggu Capsule group).The rats were orally administered drugs according to their body weight and were continuously treated for three months.After the treatment,the remaining five rats in each group were euthanized under anesthesia to collect the fifth lumbar vertebrae and right mandibles.The lumbar vertebrae and mandibles collected from the nine groups were subjected to Micro-CT analysis to measure bone density and bone morphometric parameters.Paraffin sections were prepared,and immunohistochemical methods were used to detect the expression of SDF-1/CXCR4 axis key factors and bone differentiation-specific markers RUNX2,OCN,OPN,and COLI in bone marrow mesenchymal stem cells from the lumbar vertebrae and mandibles of the rats.Results1 The effect of Qianggu Capsule on lumbar vertebrae in ovariectomized OP rats1.1 The effect of Qianggu Capsule on the microstructure of lumbar vertebrae in OVX OP ratsCompared with the baseline group,the blank group(3 months)and the sham surgery group(3 months)showed a significant increase in BMD,BV/TV and Tb.N of the lumbar vertebrae(P<0.05),while the OVX group(3 months)showed a significant decrease in BMD,BV/TV and Tb.N of the lumbar vertebrae(P<0.01),while the value of Tb.Sp was significantly increased(P<0.01).Moreover,the BMD,BV/TV and Tb.N of the lumbar vertebrae in the OVX group(3 months)was significantly lower than that in the sham surgery group(3 months)(P<0.01),while the value of Tb.Sp was significantly increased(P<0.01).Compared with the sham surgery group(6 months),the BMD and BV/TV of the lumbar vertebrae in the OVX group(6 months),positive control group,and Qianggu Capsule group all showed a significant decrease(P<0.05),while the value of Tb.Sp was significantly increased(P<0.01).Compared with the OVX group(6 months),the BMD,BV/TV and Tb.N of the lumbar vertebrae in the positive control group and Qianggu Capsule group showed a trend of increase,while the value of Tb.Sp was significantly decreased(P<0.01).The difference in Tb.Th of the lumbar vertebrae among the groups of rats was not statistically significant(P>0.05).1.2 Effects of Qianggu Capsule on the SDF-1/CXCR4 axis in the lumbar vertebrae of ovariectomized OP rats Compared with the sham group(6 months),the SDF-1 and CXCR4 protein expression in BMSCs of the lumbar vertebrae of rats in the OVX group(6 months)was significantly increased(P<0.01).Compared with the OVX group(6 months),the expression SDF-1 and CXCR4 in the positive control group and Qianggu Capsule group was decreased,but still significantly higher than that in the sham group(6 months)(P<0.01).1.3 Effect of Qianggu Capsule on the expression of osteogenic differentiation-related factors RUNX2,OCN,OPN,and COLI proteins in lumbar vertebrae BMSCs of ovariectomized OP ratsThe expression level of RUNX2,OCN,OPN and COLI protein in lumbar vertebral BMSCs of the OVX group(6 months)was significantly lower than that of the sham group(6 months)(P<0.05),while the expression levels of RUNX2,OCN and COLI protein in the positive control group and Qianggu Capsule group were significantly higher than that of the OVX group(6 months)(P<0.05),but the expression level of RUNX2 and OCN protein in the positive control group was still significantly lower than that of the sham group(6 months)(P<0.05).2 The effect of Qianggu Capsule on the mandible of ovariectomized OP rats.2.1 The effect of Qianggu Capsule on the microstructure of the mandible in ovariectomized OP ratsCompared with the baseline group,the BMD,BV/TV and Tb.Th of the mandible in the blank group(3 months)and the sham group(3 months)rats were significantly increased(P<0.05),while the BMD of the mandible in the OVX group(3 months)was significantly decreased(P<0.05)and the value of Tb.N and Tb.Sp were decreased.The BMD,BV/TV and Tb.Th of the mandible in the OVX group(3 months)was lower than that in the sham group(3 months).Compared with the sham group(6 months),the BMD,BV/TV,Tb.Th and Tb.N of the mandible in the OVX group(6 months)were significantly decreased(P<0.05),while the value of Tb.Sp was significantly increased(P<0.01).Compared with the OVX group(6 months),the BMD,BV/TV and Tb.Th of the mandible in the positive control group and the Qianggu Capsule group were significantly increased(P<0.05),while the value of Tb.Sp was significantly decreased(P<0.05).2.2 Effects of Qianggu Capsule on the SDF-1/CXCR4 axis in the mandible of ovariectomized OP ratsCompared to the sham group at 6 months,the expression level of SDF-1 and CXCR4 protein in mandibular BMSCs was significantly increased in the OVX group(P<0.01);compared to the OVX group at 6 months,the SDF-1 and CXCR4 level was significantly reduced in the positive control group and Qianggu Capsule group(P<0.05).2.3 Effects of Qianggu Capsule on the expression of osteogenic differentiation-related factors RUNX2,OCN,OPN,and COLI proteins in mandibular BMSCs of ovariectomized OP ratsCompared with the sham surgery group(6 months),the expression levels of RUNX2,OCN,OPN and COLI protein in mandibular BMSCs were significantly decreased in the OVX group(6 months),positive control group,and Qianggu Capsule group(P<0.05).Compared with the OVX group(6 months),the expression levels of RUNX2,OCN and COLI protein in the positive control group and Qianggu Capsule group were significantly increased(P<0.05).3 Differences in the effects of Qianggu Capsule on "teeth-bone"changes in ovariectomized OP ratsIn the OVX group of rats,the BV/TV and BMD changes in the lumbar vertebrae and mandible are less than 0,indicating a decrease in bone mass.Additionally,the absolute values of BV/TV and BMD changes in the lumbar vertebrae are smaller than those in the mandible in the OVX group.Both the positive control group and the Qianggu Capsule group show that the absolute values of BV/TV and BMD changes in the lumbar vertebrae are smaller than those in the corresponding groups’ mandible.In the OVX group of rats,the Tb.N changes in the lumbar vertebrae and mandible are less than 0,while Tb.Sp is greater than 0,indicating the occurrence of osteoporosis in both the lumbar vertebrae and mandible.Moreover,the absolute values of Tb.N and Tb.Sp changes in the lumbar vertebrae are smaller than those in the mandible in the OVX group.Both the positive control group and the Qianggu Capsule group show that the absolute values of Tb.N and Tb.Sp changes in the lumbar vertebrae are smaller than those in the corresponding groups’ mandible.Conclusion(1)Bilateral ovariectomy can cause a decrease in bone density,changes in bone morphometry,and manifestations of osteoporosis such as reduced trabecular number and increased trabecular separation in the lumbar vertebrae and mandible of rats.The Qianggu Capsule,a traditional Chinese medicine for tonifying the kidneys,can improve bone microstructure,increase lumbar vertebrae and mandible bone density,and treat ovariectomy-induced osteoporosis.(2)Bilateral ovariectomy can significantly increase the expression levels of SDF-1 and its receptor CXCR4 in BMSCs in the lumbar vertebrae and mandible of rats,and significantly decrease the expression of osteogenic differentiation-specific factors RUNX2,OPN,OCN,and COLI.After intervention with the Qianggu Capsule,the levels of SDF-1 and CXCR4 in castrated rats decreased,and the expression of osteogenic factors correspondingly increased.It is speculated that the main active ingredient of the Qianggu Capsule,total flavonoids of Epimedium,activates the SDF-1/CXCR4 axis to promote BMSC homing and osteogenesis,thereby improving ovariectomy-induced bone metabolism disorders and exerting a bone-protective effect.(3)There is a certain difference in the therapeutic effect of Qianggu Capsule on the lumbar vertebrae and mandible,with a more significant effect on the lumbar vertebrae.The differential changes in the intervention of kidney-tonifying Chinese herbal medicine on the lumbar vertebrae and mandible suggest a certain correlation between the therapeutic effect of Qianggu Capsule on osteoporosis and the classic Chinese medicine theory of "the kidney governs bone and generates marrow" and "teeth are the surplus of bones."... |
PDF Full Text Request