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The effect of amygdalin joint hydroxysafflor yellow A (HSYA) on the endplate chondrocytes derived from intervertebral discs of rats induced by IL-1beta and the possible mechanism were studied and explored. Chondrocytes were obtained from endplate of one-month SD rat intervertebral discs and cultured primary endplate chondrocytes. After identification, they were divided into normal group, induced group, amygdalin group, HSYA group and combined group. CCK-8 kit was adopted to detect the proliferation of the endplate chondrocytes. FCM was measured to detect the apoptosis. Real-time PCR method was adopted to observe the mRNA expression of Aggrecan, Col 2 alpha1, Col 10 alpha1, MMP-13 and the inflammatory cytokines IL-1beta. The protein expression of Col II, Col X was tested through immunofluorescence. Compared with the normal group, the proliferation of the endplate chondrocytes decreased while the apoptosis increased (P < 0.05). With down regulation of the mRNA expressions of Aggrecan, Col 2 alpha1 and up regulation of the mRNA expressions of Col 10 alpha1, MMP-13, IL-1beta (P < 0.05), the protein expression of Col II decreased while the protein expression of Col X increased. Compared with the induced group, amygdalin group, HSYA group, the combined group could inhibit the apoptosis and promote the proliferation (P < 0.05). They could increase the mRNA expressions of Aggrecan and Col 2 alpha1 while decrease the mRNA expressions of Col 10 alpha1, MMP-13 and IL-1beta (P < 0.05). They could also enhance the protein expression of Col II while reduce the protein expression of Col X. The effect of the combined group was significantly better than that of amygdalin and HSYA. Amygdalin joint HSYA could inhibit the degeneration of the endplate chondrocytes derived from intervertebral discs of rats induced by IL-1beta and better than the single use of amygdalin or HSYA.

Objective: To observe effects of removing arms and ovarian on lumbar intervertebral disc and vertebral bone mineral density (BMD) by establishing rat model of lumbar intervetebral disc degeneration (IDD) with kidney deficiency, and to explore internal mechanism of disc degeneration, relationship between disc degeneration and osteoporosis., Methods: Thirty Sprague-Dawley female rats aged one month were randomly divided into control group, lumbar IDD group and lumbar IDD with kidney deficiency group (combined group), 10 rats in each group. Lumbar IDD group removed double arms, lumbar IDD with kidney deficiency group removed double arms after 3 months, both ovaries were removed. Vertebral bone mineral density were observed by Micro-CT scan; morphological changes were tested by safranine O-fast green staining; II, X collagen protein expression in the intervertebral disc were obsevered by immunohistochemistry; extracellular matrix gene expression were obsevered by real-time polymerase chain reaction (RT-PCR), in order to evaluate the effects of removed of forelimbs and double ovarian on degeneration and vertebral bone mineral density of intervertebral disc., Results: Micro-CT scan showed osteoporosis in kidney deficiency group was obviously worse than other two groups; safranine O-fast green staining showed that intervertebral space became narrowed, intervertebral disc tissue degenerated obviously, chondral palte was underdeveloped in kidney deficiency group; immunohistochemistry showed that X collagen expression increased, type II collagen expression decreased in kidney deficiency group; RT-PCR showed that type II collagen expression in lumbar IDD group and kidney deficiency group was lower than control group, and had statistical meaning among three groups (P=0.000, P=0.000); Age 1 in lumbar IDD group and kidney deficiency group was lower than control group, and had statistical meaning among three groups (P=0.000, P= 0.000); while type X collagen expression was higher than control group, but no significant meaning; MMP-13 in lumbar IDD group and kidney deficiency group was higher than control group, with significant meaning compared among three groups (P= 0.000, P=0.000); aggrecanase-2 in lumbar IDD group and kidney deficiency group was higher than control group, with significant meaning compared among three groups (P=0.006, P=0.008)., Conclusion: Rats model of lumbar disc degeneration established by removed forelimbs and ovariectomized can occure "bone like"--osteoporosis, which is similar with clinical kidney lumbar disc degeneration in tissue morphology, molecular cell biology expression.

Icariin has been mostly reported to enhance bone fracture healing and treat postmenopausal osteoporosis in ovariectomized animal model. As another novel animal model of osteoporosis, there is few publication about the effect of Icariin on osteoprotegerin-deficient mice. Therefore, the goal of this study is to find the effect on bone formation and underlying mechanisms of Icariin in osteoprotegerin (OPG) knockout (KO) mice. We found that Icariin significantly stimulated new bone formation after local injection over the surface of calvaria at the dose of 5 mg/kg per day. With this dose, Icariin was also capable of significantly reversing OPG-deficient-induced bone loss and bone strength reduction. Real-time PCR analysis showed that Icariin significantly upregulated the expression of BMP2, BMP4, RUNX2, OC, Wnt1, and Wnt3a in OPG KO mice. Icariin also significantly increased the expression of AXIN2, DKK1, TCF1, and LEF1, which are the direct target genes of β -catenin signaling. The in vitro studies showed that Icariin induced osteoblast differentiation through the activation of Wnt/ β -catenin-BMP signaling by in vitro deletion of the β -catenin gene using β -catenin(fx/fx) mice. Together, our findings demonstrate that Icariin significantly reverses the phenotypes of OPG-deficient mice through the activation of Wnt/ β -catenin-BMP signaling.

Icariin (ICA) is an active component of Herba Epimedium effective in preventing osteoporosis. Bone mesenchymal stem cells (bMSCs) are an important target by which ICA promotes osteogenesis. However, its molecular mechanisms are poorly defined. In the present study, we induced osteoporosis in rats by corticosterone (CORT) and ovariectomy (OVX), treated both with ICA for 2 weeks or 3 months. As results, both models displayed bone loss tendency within 2 weeks and a significant bone loss after 3 months. ICA promoted bMSCs diffenentiation from CORT rat, and increased the secretion of osteocalcin, collagen I, runt-related transcription factor 2 in OVX model. Gene profile revealed a marked shift of gene expression by ICA, with much more significance in CORT rats. These potential molecular targets were involved in cell communication, adhesion, cycle and cytokines secretion. But very few genes overlapped in these two models, suggesting the effects and molecular mechanisms of ICA on osteoporosis might be pathogenesis-dependent. However, the Notch signaling pathway was common in both models, and should be paid close attention to for further study.

Objective: To investigate the effects of serum containing Yiqi Huayu Bushen Recipe, a compound Chinese herbal medicine, on expressions of aggrecan and type X collagen mRNAs of the degenerated intervertebral disc cells in patients with cervical spondylotic myelopathy., Methods: Six inpatients diagnosed as cervical spondylotic myelopathy and undergoing cervical decompression and fusion surgery to remove the degenerated disc tissue were enrolled in this study. Primary intervertebral disc cells were isolated using small tissue mass method. Serum containing Yiqi Huayu Bushen recipe were obtained after SD rats were fed with Yiqi Huayu Bushen recipe for 3 days. MTT method was used to detect proliferation of the first-passage cells isolated from degenerated intervertebral discs and growth curve was drawn. In the following tests, the first-passage cells were cultured with the sera containing drugs and sodium chloride (NaCl), respectively. Proliferation of intervertebral disc cells was detected by MTT method and expressions of aggrecan and type X collagen mRNAs were detected by real-time polymerase chain reaction., Results: The growth curve of the first-passage cells of the degenerated intervertebral discs showed that the time of cell proliferation peak was the sixth day. Compared with the same concentrations of NaCl solution, serum containing Yiqi Huayu Bushen Recipe at 5%, 10% and 20% promoted cell proliferation and expression of aggrecan mRNA and decreased expression of type X collagen mRNA (P<0.01). 10% serum containing Yiqi Huayu Bushen recipe was more effective than 5% and 20%., Conclusion: Yiqi Huayu Bushen Recipe inhibits degeneration of the intervertebral disc cells by regulating collagen and proteoglycan expressions.

Objective: To observe the effects of icariin, psoralen and oleanolic acid, the three active ingredients of Yinyanghuo (Herba Epimedii Brevicornus), Buguzhi (Fructus Psoraleae) and Nuzhenzi (Fructus Ligustri Lucidi), respectively, on gene expression profile of bone marrow stromal cells (BMSCs) from rats with corticosterone-induced osteoporosis., Methods: Fifty Sprague-Dawley rats were randomly divided into normal control group, untreated group, icariin group, psoralen group and oleanolic acid group (the last three groups are called treatment groups). Rats in untreated group and treatment groups were subcutaneously injected with corticosterone once a day for 14 d while rats in normal control group were injected isodose of olive oil. Rats in the treatment groups were intragastrically administered with icarrin, or psoralen, or oleanolic acid at 20 mg/(kg·d), respectively, 5 d prior to modeling for 19 d. The body weight of rats was recorded on the 1st, 4th, 7th, 11th, and 14th day after modeling, respectively. All rats were sacrificed and the fourth lumbar vertebrae were harvested for micro-CT scanning to evaluate bone mass. BMSCs were obtained ex vivo by the methods of bone marrow adherent culture. The mRNAs of BMSCs were detected by using gene chip technique on the 7th day of culture., Results: There was no significant difference in body weight of rats between untreated group and treatment groups. Micro-CT showed no significant difference in lumbar vertebral morphometry between the untreated and the normal control, or the untreated and treatment groups. In microarray, icariin, psoralen and oleanolic acid changed expressions of 11, 12 and 15 genes compared to the normal level, respectively. These three active ingredients all acted on 5 genes involving osteoblast differentiation, cell cycle regulation, cell metabolism and Notch signal pathway., Conclusion: Traditional Chinese herbal drugs with the effect of tonifying the kidney may promote BMSCs to differentiate into osteoblasts by regulating BMSCs cycles and cell metabolism, and show therapeutic effect on osteoporosis. However, the exact mechanisms need to be further studied.

Astragaloside IV (ASI), a pure compound derived from Radix Astragali, is commonly used in degenerative bone diseases such as osteoporosis. Our previous study identified in vivo the osteogenetic effect of Fu Fang Qi She Pills (FFQSP), a Chinese herbal formula containing Radix Astragali from which ASI was extracted. In this study, we investigated the osteogenetic effects of ASI under the conditions of centrifugating pressure on OCT-1 cells. These preosteoblasts were grown in 3D-culture, and treated with ASI at 50 micromol/l with centrifugation at 200 rpm, 500 rpm for 3 and 5 days. Morphocytological examination, morphometry of alkaline phosphatases (ALP) staining was performed. Expression of type I collagen (Col I) was detected by immunocytochemistry assays. ALP, Col1a2, Osteocalcin (OC), and runt-related transcription factor-2 (Runx2) mRNA expression were determined via real-time PCR. The results showed ASI plus 500 rpm for 3 days and ASI plus 200 rpm for 5 days significantly induced osteogenesis related protein and gene expression. We concluded that ASI would promote osteogenesis when cells of preosteoblast OCT-1 were subjected to proper centrifugating pressure and a pertinent period of time.

Objective: To observe the effects of Qishe Pill, a compound traditional Chinese herbal medicine, on lumbar vertebral bone formation induced by long-time upright posture in rats and to investigate the potential mechanism., Methods: Thirty SD rats were randomly divided into normal control group, untreated group and Qishe Pill group. The rats in normal control group received no treatment and were raised in normal cages. The rats in untreated group and Qishe Pill group were cut off forelimbs by operation so as to be forced to adopt an upright posture for 8 months to induce hyperostosis. Rats in the Qishe Pill group were intragastrically administered with Qishe Pill at a dose of 5 g/(kg x d) for 1 month. All rats were sacrificed at the 9th month after surgery and all lumbar vertebrae were harvested for detection. Safranin O/fast green staining and picrosirius red staining were used to observe pathological changes. Expressions of type I collagen (ColI), type X collagen (ColX), vascular endothelial growth factor (VEGF) and transforming growth factor beta1 (TGF-beta1) in the 5th lumbar vertebra (L(5)) were detected by immunohistochemical method. Expressions of type I collagen alpha2 (Col1alpha2), type X collagen alpha1 (Col10alpha1), TGF-beta1, and VEGF and runt-related transcription factor 2 (Runx2) mRNAs in L(1)-L(3) were detected by real-time fluorescent quantitation reverse transcription-polymerase chain reaction., Results: Safranin O/fast green staining showed that in the untreated group, non-matrix components increased at marginal lumbar vertebra and intervertebral disc junction, and hyperostosis appeared. However, no obvious change was observed in the normal control group. Non-matrix components decreased at the same location in Qishe Pill group as compared with the untreated group. Picrosirius red staining showed compact collagens at marginal lumbar vertebra and intervertebral disc junction in the normal control group, however, Col I and ColX increased at the same location in the untreated group. In the Qishe Pill group, it showed more compact collagens, especially ColI. Compared with normal control group, expressions of ColX, VEGF and TGF-beta1 were increased at marginal lumbar vertebra and intervertebral disc junction in the untreated group. ColX and VEGF expressions decreased in the Qishe Pill group as compared with the untreated group. Col10alpha1 and Runx2 mRNA expressions were down-regulated by Qishe Pill (P<0.01)., Conclusion: Qishe Pill may delay hyperostosis at marginal lumbar vertebra and intervertebral disc junction, which may be related to reducing type X collagen and Runx2 expressions.