A novel therapy for fracture healing by increasing lymphatic drainage.

Background: The musculoskeletal system contains an extensive network of lymphatic vessels. Decreased lymph flow of the draining collecting lymphatics usually occurs in clinic after traumatic fractures. However, whether defects in lymphatic drainage can affect fracture healing is unclear. Methods: To investigate the effect of lymphatic dysfunction on fracture healing, we used a selective VEGFR3 tyrosine kinase inhibitor to treat tibial fractured mice for 5 weeks versus a vehicle-treated control. To ensure successfully establishing deceased lymphatic drainage model for fractured mice, we measured lymphatic clearance by near infrared indocyanine green lymphatic imaging (NIR-ICG) and the volume of the draining popliteal lymph nodes (PLNs) by ultrasound at the whole phases of fracture healing. In addition, hindlimb edema from day 0 to day 7 post-fracture, pain sensation by Hargreaves test at day 1 post-fracture, bone histomorphometry by micro-CT and callus composition by Alcian Blue-Hematoxylin/Orange G staining at day 14 post-fracture, and bone quality by biomechanical testing at day 35 post-fracture were applied to evaluate fracture healing. To promote fracture healing via increasing lymphatic drainage, we then treated fractured mice with anti-mouse podoplanin (PDPN) neutralizing antibody or isotype IgG antibody for 1 week to observe lymphatic drainage function and assess bone repair as methods described above. Results: Compared to vehicle-treated group, SAR-treatment group significantly decreased lymphatic clearance and the volume of draining PLNs. SAR-treatment group significantly increased soft tissue swelling, and reduced bone volume (BV)/tissue volume (TV), trabecular number (Tb.N), woven bone and biomechanical properties of fracture callus. In addition, anti-PDPN treated group significantly reduced the number of CD41+ platelets in PLNs and increased the number of pulsatile lymphatic vessels, lymphatic clearance and the volume of PLNs. Moreover, anti-PDPN treated group significantly reduced hindlimb edema and pain sensation and increased BV/TV, trabecular number (Tb.Th), woven bone and biomechanical properties of fracture callus. Conclusions: Inhibition of proper lymphatic drainage function delayed fracture healing. Use of a anti-PDPN neutralizing antibody reduced lymphatic platelet thrombosis (LPT), increased lymphatic drainage and improved fracture healing. The translational potential of this article: (1) We demonstrated lymphatic drainage function is crucial for fracture healing. (2) To unblock the lymphatic drainage and prevent the risk of bleeding and mortality by blood thinner, we demonstrated PDPN neutralizing antibody is a novel and safe way forward in the treatment of bone fracture healing by eliminating LPT and increasing lymphatic drainage.

A Naringin- and Icariin-Contained Herbal Formula, Gushukang, Ameliorated Aged Osteoporosis of Aged Mice with High Calcium Intake.

Traditional herbal formula Gushukang (GSK) was clinically applied to treat primary osteoporosis and showed osteoprotective effect in ovariectomized rodent animals and regulatory action on calcium transporters. This study aimed to determine if GSK could ameliorate aged osteoporosis by modulating serum level of calciotropic hormones and improving calcium balance. 18-month-old male mice were orally administered with either GSK (0.38[Formula: see text]g/kg body weight) or calcitriol (1[Formula: see text][Formula: see text]g/kg body weight) combined with high calcium diet (HCD, 1.2% Ca) for 60 days. The aged mice fed with normal calcium diet (NCD, 0.6% Ca) were a negative control. Trabecular bone and cortical bone properties as well as calcium balance were determined. Treatment with GSK significantly increased 25(OH)D and 1,25-(OH)2D levels in serum, moreover, it markedly attenuated trabecular bone micro-architectural deteriorations and elevated trabecular bone mass as well as strengthened cortical bone mechanical properties shown by the increase in maximal bending load and elastic modulus. Calcium balance, including urinary Ca excretion, fecal Ca level and net calcium retention, was remarkably improved by GSK, which up-regulated TRPV6 expression in duodenum and TRPV5 expression in kidney and down-regulated claudin-14 expression in duodenum and kidney. Additionally, 1-OHase and 24-OHase expression was significantly decreased (vs. NCD group) and increased (vs. HCD group), respectively, in kidney of GSK- and calcitriol-treated mice. Taken together, this study demonstrated the ameliorative effects of Gushukang on aged osteoporosis by effectively stimulating vitamin D production and improving calcium balance of aged mice with high dietary calcium supplement.

The Natural Compound Notopterol Binds and Targets JAK2/3 to Ameliorate Inflammation and Arthritis.

The traditional Chinese medicinal herb Notopterygium incisum Ting ex H.T. Chang has anti-rheumatism activity, and a mass spectrometry assay of patients' serum after administration of the herb revealed that notopterol is the most abundant component enriched. However, the functions of notopterol and its molecular target in rheumatoid arthritis (RA) treatment remain unknown. Here, we show in different RA mouse strains that both oral and intraperitoneal administration of notopterol result in significant therapeutic effects. Mechanistically, notopterol directly binds Janus kinase (JAK)2 and JAK3 kinase domains to inhibit JAK/signal transducers and activators of transcription (JAK-STAT) activation, leading to reduced production of inflammatory cytokines and chemokines. Critically, combination therapy using both notopterol and tumor necrosis factor (TNF) blocker results in enhanced therapeutic effects compared to using TNF blocker alone. We demonstrate that notopterol ameliorates RA pathology by targeting JAK-STAT signaling, raising the possibility that notopterol could be effective in treating other diseases characterized by aberrant JAK-STAT signaling pathway.

Alleviation of Synovial Inflammation of Juanbi-Tang on Collagen-Induced Arthritis and TNF-Tg Mice Model.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is primarily characterized by synovial inflammation. In this study, we found that a traditional Chinese decoction, Juanbi-Tang (JBT), JBT attenuated the symptoms of collagen-induced arthritis (CIA) mice and in tumor necrosis factor transgenic (TNF-Tg) mice by attenuating the arthritis index and hind paw thickness. According to histopathological staining of ankle sections, JBT significantly decreased the area of inflammation and reduced bone destruction of ankle joints in both these two types of mice. Moreover, decreased tartaric acid phosphatase-positive osteoclasts were observed in the JBT group compared with those found in the control group. We also revealed that JBT suppressed monocytes and T cells as well as the production of CCL2, CCR6, and CXCR3 ligands. We next used high-performance liquid chromatography to investigate the components and pharmacological properties of this classical herbal medicine in traditional Chinese medicine. Based on network pharmacology, we performed computational prediction simulation of the potential targets of JBT, which indicated the NF-kappa B pathway as its target, which was confirmed in vitro. JBT suppressed the production of pro-inflammatory cytokines including interleukin-6 (IL-6) and IL-8, and inhibited the expression of matrix metalloproteinase 1 in fibroblast-like synoviocytes derived from RA patients (MH7A cells). Furthermore, JBT also suppressed the phosphorylation of p38, JNK, and p65 in TNF-α-treated MH7A cells. In summary, this study proved that JBT could inhibit synovial inflammation and bone destruction, possibly by blocking the phosphorylation of NF-kappa B pathway-mediated production of proinflammatory effectors.

Velvet antler polypeptide partially rescue facet joint osteoarthritis-like phenotype in adult ß-catenin conditional activation mice.

BACKGROUND: Wnt/ß-catenin signaling pathway is closely related to osteoarthritis. In our preliminary study, ß-catenin conditional activation (cAct) mice that specifically over-express ß-catenin gene in cartilage chondrocyte exhibits osteoarthritis-like phenotype in the lumbar disc and knee joint. Therefore, we used the mice to model FJ-OA and test the potential curative effect of Velvet Antler Polypeptide (VAP) on this mice model. METHODS: We tested the effect of VAP on ß-catenin conditional activation mice, and used Cre negative littermates as controls. Micro-CT, histology and histomorphometry analysis were performed to evaluate the curative effect of VAP on mice facet joint-like phenotype. Expression of ß-catenin and collagen II was detected by immunohistochemistry (IHC) and western-blot., MMP13, ADAMTS4 and ADAMTS5 was detected by immunofluorescence (IF). RT-PCR analysis was preformed to detect mRNA expression of cartilage degrading enzymes, such as MMP13, ADAMTS4 and ADAMTS5. RESULTS: Results of micro-CT (µCT) analysis showed that VAP could partially reverse lumbar disc osteophyte formation observed in ß-catenin(ex3)Col2ER mice. Histology data revealed VAP partially improved facet joint cartilage tissue invades. Histomorphometry analysis showed an increase in total cartilage area after VAP treatment. IHC show that VAP reduced ß-catenin protein levels and moderately up-regulated collagen II protein levels. RT-PCR and IF data showed that VAP down-regulated the expression of extracellular matrix synthesis (ECM) degradation enzymes MMP13, ADAMTS4 and ADAMTS5. CONCLUSION: Taken together, VAP may modulate ECM by inhibits MMP13, ADAMTS4 and ADAMTS5 via Wnt /ß-catenin signaling pathway. Velvet Antler Polypeptide may be a potential medicine for FJ-OA.

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments.

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

The systemic bone protective effects of Gushukang granules in ovariectomized mice by inhibiting osteoclastogenesis and stimulating osteoblastogenesis.

Primary osteoporosis (POP), which is caused by unbalanced bone remodeling, leads to significant economic and societal burdens globally. Gushukang (GSK) granule serves as one commonly used prescription for POP in Traditional Chinese Medicine (TCM). The present study aimed to clarify the exact roles of GSK in bone remodeling with in vivo and in vitro assays. Here we showed that GSK prevented bone loss and the alternations of osteoporotic bone parameters as well as the decreased density of osteoclast in ovariectomized (OVX) mice. GSK inhibited receptor activator for nuclear factor-κ B Ligand (RANKL)-activated osteoclastogenesis in bone marrow macrophages (BMMs). At the molecular levels, GSK inhibited the expression of nuclear factor of activated T cells cytoplasm 1(NFATc1) and c-Fos, two master regulators of osteoclastogenesis. GSK also inhibited bone resorbed genetic expression of matrix metalloproteinase 9 (MMP9), cathepsin K (Ctsk), TRAP and carbonic anhydrase II (Car2). Meanwhile, GSK stimulated osteoblastogenesis from bone primary mesenchymal stem cells (MSCs) and enhanced the expression of Osteirx, and Runx2. GSK also stimulated the expression of Col-1, Osteocalcein and alkaline phosphatase (ALP). Our investigation established the systemic bone protective effects of GSK by suppressing osteoclastogenesis and stimulating osteoblastogenesis and laid bases for new drugs discovery in treating POP.

Effect of the water fraction isolated from Fructus Ligustri Lucidi extract on bone metabolism via antagonizing a calcium-sensing receptor in experimental type 1 diabetic rats.

Our previous studies have demonstrated that the extract of Fructus Ligustri Lucidi (FLL) can maintain in vivo calcium homeostasis in aged and ovariectomized rats. This study was designed to elucidate the action of water fraction isolated from the FLL extract on bone metabolism and a calcium-sensing receptor (CaSR) in parathyroid glands and kidneys of diabetic rats. The streptozotocin-induced diabetic rats were treated with vehicle, FLL extract, and the water fraction (WF) isolated from the FLL extract for 4 weeks. Treatment with WF dramatically increased the serum levels of both calcium and parathyroid hormone and reduced urinary calcium excretion in diabetic rats as well as improved the pathological changes of trabecular bone as shown by the increased BA/TA, BMD/BV, and BV/TV. The mRNA expression of the calcium-binding protein 9k and protein expression of a vitamin D receptor (VDR) and plasma membrane Ca-ATPase in duodenum were significantly increased in diabetic rats after treatment with WF, which reduced the expression of CaSR in parathyroid gland and kidney as well as inhibited the up-regulation of VDR and 25-hydroxyvitamin D-24 hydroxylase expressions in the kidney of diabetic rats. This study reveals that the water fraction may be an active component of the FLL extract that exerts beneficial effects on improving bone metabolism via regulating vitamin D metabolism in kidney and vitamin D-dependent calcium transporters in duodenum as well as modulating the expression of CaSR in the parathyroid gland and kidneys.

Osteoprotective effects of osthole in a mouse model of 5/6 nephrectomy through inhibiting osteoclast formation.

The present study aimed to investigate the effects of osthole on osteoclast formation and bone loss in a mouse model of 5/6 nephrectomy. The mice in control and osthole groups were treated 1 month following 5/6 nephrectomy with either a placebo or osthole, respectively. At 2 months post­nephrectomy, the L4 vertebrae were harvested. The bone mineral density (BMD) of cancellous bone was measured using micro­CT and tartrate­resistant acid phosphatase (TRAP) staining was performed to evaluate osteoclast formation. Immunohistochemistry staining and reverse transcription­quantitative polymerase chain reaction were performed to detect the expression of nuclear factor of activated T­cells, cytoplasmic­1 (NFATc­1), c­Fos, cathepsin K, Trap, matrix metalloproteinase 9 (Mmp9), osteoprotegerin (Opg) and receptor activator for nuclear factor­κB ligand (Rankl). Bone marrow cells were cultured with osthole, and osteoclast formation was shown by TRAP staining. Primary calvaria osteoblasts were cultured with osthole, and expression levels of Opg and Rankl were detected. Compared with the sham group, the BMD of mice in model group was significantly reduced. The numbers of osteoclasts and the expression levels of NFATc­1, c­Fos, cathepsin K and Mmp9 were significantly increased. Compared with the control group, the mice in the osthole group exhibited increased BMD of the L4 vertebrae, a reduction in osteoclast numbers and decreased expression levels of NFATc­1, c­Fos, cathepsin K and Mmp9. In vitro experiments also showed that osteoclast formation was decreased following treatment with osthole. Osteoprotegerin (Opg)/receptor activator for nuclear factor­κB ligand (Rankl) was upregulated by osthole treatment in the L4 vertebrae and in primary cultures of calvarial osteoblasts. Osthole inhibited osteoclast formation and partially reversed the bone loss induced by 5/6 nephrectomy in mice through the upregulation of OPG/RANKL.

Er-Xian Decoction Stimulates Osteoblastic Differentiation of Bone Mesenchymal Stem Cells in Ovariectomized Mice and Its Gene Profile Analysis.

We studied the bone mesenchymal stem cells (bMSCs) and gene profiles regulated by Er-Xian Decoction (EXD), a traditional Chinese herbal formula widely used for postmenopausal osteoporosis treatment. Six-month-old female Imprinting Control Region mice that underwent ovariectomy were treated with EXD. After 3 months, bone mass was evaluated by µCT and histological and immunohistochemical detection. The self-renewal and differentiation capacities of bMSCs were evaluated by colony-forming unit-fibroblastic, colony-forming unit-adipocyte, and alkaline phosphatase staining. In addition, the expression of 26991 genes of bMSCs ex vivo at 2 weeks after EXD-treatment or of bMSCs in vitro after exposure to conditioned serum from EXD-treated rats was measured and analyzed using NimbleGen Gene Expression Profiling and Cluster and pathway analysis. EXD treatment increased bone mass, elevating osteocalcin protein levels in vivo and facilitating the self-renewal and osteoblastic differentiation of bMSCs ex vivo. EXD rescued several gene expressions that were dysregulated by OVX. These genes overlapped and their functions were involved in ten pathways between ex vivo and in vitro experiments. EXD exerts an osteogenic effect on bMSCs in OVX induced osteoporotic mice. Our results contribute to further study of its molecular mechanism and traditional use in the treatment of postmenopausal osteoporosis.

Molecular mechanisms of Polyphyllin I-induced apoptosis and reversal of the epithelial-mesenchymal transition in human osteosarcoma cells.

Osteosarcoma is a most common highly malignant bone tumor in children and adolescents. Polyphyllin I (PPI) is an ethanol extraction from Paris polyphylla Smith var.yunnanensis (Franch.) Hand.-Mazz, which belongs to antipyretic-detoxicate family and has been used as a natural medicine in the treatment of infectious disease and cancer in China for centuries. The proteasome activity inhibitory and anti-osteosarcoma effects of PPI have not been known. Here we found PPI exhibited a selective inhibitory effect on proteasomal chymotrypsin (CT)-like activity, both in purified human proteasome and in cultured osteosarcoma cellular proteasome, and caused an accumulation of ubiquitinated proteins. PPI also inhibited viability, proliferation, migration, and invasion of MG-63, Saos-2, and U-2 OS osteosarcoma cells and resulted in S phase arrest and apoptosis. Furthermore, we explored the molecular targets involved. Exposure of osteosarcoma cells to PPI caused an inactivation of the intrinsic nuclear factor κB (NF-κB) and activation of unfolded protein response (UPR)/endoplasmic reticulum (ER) stress signaling cascade in osteosarcoma cells, followed by down-regulation of anti-apoptotic proteins, with up-regulation of pro-apoptotic proteins. We also demonstrated down-regulation of c-Myc, Cyclin B1, Cyclin D1, and CDK1, which are involved in the cell cycle and growth. Finally, we identified down-regulation of Vimentin, Snail, Slug, and up-regulation of E-cadherin, which are integral proteins involved in epithelial-mesenchymal transition (EMT). Taken together, our data provide insights into the mechanism underlying the anticancer activity of PPI in human osteosarcoma cells.

Protective effect of ligustrazine on lumbar intervertebral disc degeneration of rats induced by prolonged upright posture.

Most chronic low back pain is the result of degeneration of the lumbar intervertebral disc. Ligustrazine, an alkaloid from Chuanxiong, reportedly is able to relieve pain, suppress inflammation, and treat osteoarthritis and it has the protective effect on cartilage and chondrocytes. Therefore, we asked whether ligustrazine could reduce intervertebral disc degeneration. To determine the effect of ligustrazine on disc degeneration, we applied a rat model. The intervertebral disc degeneration of the rats was induced by prolonged upright posture. We found that pretreatment with ligustrazine for 1 month recovered the structural distortion of the degenerative disc; inhibited the expression of type X collagen, matrix metalloproteinase (MMP)-13, and MMP3; upregulated type II collagen; and decreased IL-1 ß , cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) expression. In conclusion, ligustrazine is a promising agent for treating lumbar intervertebral disc degeneration disease.

Icariin Augments Bone Formation and Reverses the Phenotypes of Osteoprotegerin-Deficient Mice through the Activation of Wnt/ ß -Catenin-BMP Signaling.

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 ß -catenin signaling. The in vitro studies showed that Icariin induced osteoblast differentiation through the activation of Wnt/ ß -catenin-BMP signaling by in vitro deletion of the ß -catenin gene using ß -catenin(fx/fx) mice. Together, our findings demonstrate that Icariin significantly reverses the phenotypes of OPG-deficient mice through the activation of Wnt/ ß -catenin-BMP signaling.

[Effects of Chinese herbal medicine Yiqi Huayu Bushen Recipe on expressions of aggrecan and type X collagen mRNAs in cells from degenerated human intervertebral discs].

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.

The osteogenetic effect of astragaloside IV with centrifugating pressure on the OCT-1 cells.

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.

[Effects of active ingredients in three kidney-tonifying Chinese herbal drugs on gene expression profile of bone marrow stromal cells from a rat model of corticosterone-induced osteoporosis].

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.

[Effects of Qishe Pill on vertebral hyperostosis induced by upright posture in rats].

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.