Integration of single-cell sequencing and bulk RNA-seq to identify and develop a prognostic signature related to colorectal cancer stem cells.

The prognosis for patients with colorectal cancer (CRC) remains worse than expected due to metastasis, recurrence, and resistance to chemotherapy. Colorectal cancer stem cells (CRCSCs) play a vital role in tumor metastasis, recurrence, and chemotherapy resistance. However, there are currently no prognostic markers based on CRCSCs-related genes available for clinical use. In this study, single-cell transcriptome sequencing was employed to distinguish cancer stem cells (CSCs) in the CRC microenvironment and analyze their properties at the single-cell level. Subsequently, data from TCGA and GEO databases were utilized to develop a prognostic risk model for CRCSCs-related genes and validate its diagnostic performance. Additionally, functional enrichment, immune response, and chemotherapeutic drug sensitivity of the relevant genes in the risk model were investigated. Lastly, the key gene RPS17 in the risk model was identified as a potential prognostic marker and therapeutic target for further comprehensive studies. Our findings provide new insights into the prognostic treatment of CRC and offer novel perspectives for a systematic and comprehensive understanding of CRC development.

Serum SERCA2a levels in heart failure patients are associated with adverse events after discharge.

Calcium homeostasis imbalance is one of the important pathological mechanisms in heart failure. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a), a calcium ATPase on the sarcoplasmic reticulum in cardiac myocytes, is a myocardial systolic-diastolic Ca2 + homeostasis regulating enzyme that is not only involved in cardiac diastole but also indirectly affects cardiac myocyte contraction. SERCA2a expression was found to be decreased in myocardial tissue in heart failure, however, there are few reports on serum SERCA2a expression in patients with heart failure, and this study was designed to investigate whether serum SERCA2a levels are associated with the occurrence of adverse events after discharge in patients hospitalized with heart failure. Patients with heart failure hospitalized in the cardiovascular department of the Second Affiliated Hospital of Guangdong Medical University, China, from July 2018 to July 2019 were included in this study, and serum SERCA2a concentrations were measured; each enrolled patient was followed up by telephone after 6 months (6 ±â€…1 months) for general post-discharge patient status. The correlation between serum SERCA2a levels and the occurrence of adverse events (death or readmission due to heart failure) after hospital discharge was assessed using multiple analysis and trend analysis. Seventy-one patients with heart failure were finally included in this study, of whom 38 (53.5%) were men and 33 (46.5%) were women (All were postmenopausal women). Multiple analysis revealed no correlation between serum SERCA2a levels and the occurrence of adverse events in the total study population and in male patients, but serum SERCA2a levels were associated with the occurrence of adverse outcome events after hospital discharge in female patients (OR = 1.02, P = .047). Further analysis using a trend analysis yielded a 4.0% increase in the risk of adverse outcomes after hospital discharge for each unit increase in SERCA2a in female patients (OR = 1.04; P = .02), while no significant difference was seen in men. This study suggests that serum SERCA2a levels at admission are associated with the occurrence of post-discharge adverse events in postmenopausal female patients hospitalized with heart failure.

Metformin attenuates diabetes-induced osteopenia in rats is associated with down-regulation of the RAGE-JAK2-STAT1 signal axis.

Background: Osteopenia and fragile fractures are diabetes-associated complications. Many hypoglycemic drugs have effects on bone metabolism. Metformin, as is a prescribed medication for type 2 diabetes mellitus (T2DM), had been reported to have osteoprotective effects beyond its hypoglycemic effect, however the potential mechanism behind these effects remains unclear. In this study, we aimed to investigate the comprehensive effects of metformin on bone metabolism in T2DM rat model and elucidate the potential mechanism. Methods: Goto-Kakizaki spontaneous T2DM rats with significant hyperglycemia were treated with/without metformin for 20 weeks. Glucose tolerance was tested and all rats were weighed every two weeks. The osteoprotective effects of metformin in diabetic rats were determined by quantifying serum bone biomarkers, µ-CT imaging, histological staining, bone histomorphometry, and biomechanical properties analyses. Potential targets of metformin in the treatment of T2DM and osteoporosis were predicted using network pharmacology. The effects of metformin on mesenchymal stem cells (C3H10) cultured in high glucose medium were evaluated by CCK-8 assay, alkaline phosphatase (ALP) staining, qPCR and western blotting. Results: This study demonstrated that metformin significantly attenuated osteopenia, decreased serum glucose and glycated serum protein (GSP) levels, improved bone microarchitecture, and biomechanical properties in GK rats with T2DM. Metformin significantly increased biomarkers of bone formation, and significantly decreased muscle ubiquitin C (Ubc) expression. Network pharmacology analysis found that signal transducer and activator of transcription1 (STAT1) would be a potential target of metformin for regulating bone metabolism. Metformin increased C3H10 â€‹cell viability in vitro, alleviated ALP inhibition caused by hyperglycemia, increased the osteogenic gene expression of runt-related transcription factor 2 (RUNX2), collagen type I alpha 1 (Col1a1), osteocalcin (OCN), and ALP, while suppressing RAGE and STAT1 expression. Metformin also increased the protein expression of Osterix and decreased that of RAGE, p-JAK2, and p-STAT1. Conclusions: Our results demonstrate that metformin attenuated osteopenia and improved bone microarchitecture in GK rats with T2DM and significantly promoted stem cell osteogenic differentiation under high glucose condition. The effects of metformin on bone metabolism are closely associated with the suppression of RAGE-JAK2-STAT1 signaling axis. The translational potential of this article: Our research provides experiment evidence and potential mechanistic rationale for the use of metformin as an effective candidate for diabetes-induced osteopenia treatment.

Tubson-2 decoction ameliorates rheumatoid arthritis complicated with osteoporosis in CIA rats involving isochlorogenic acid A regulating IL-17/MAPK pathway.

BACKGROUND: Osteoporosis (OP) is considered as one of the major comorbidities of rheumatoid arthritis (RA), and is responsible for fragility fracture. However, there is currently no effective treatment for RA complicated with OP. Tubson-2 decoction (TBD), a Mongolian medicine also known as Erwei Duzhong Decoction, has been shown to exert a preventive effect on post-menopausal osteoporosis (PMOP). The preventive effects of TBD on RA-induced OP, as well as the bioactive compound responsible and the underlying mechanisms, remain to be elucidated. OBJECTIVE: To explore the effects of TBD on RA-induced OP in vivo, and to elucidate the mechanism of isochlorogenic acid A (ICA), the effective component of TBD, in vitro. METHODS: To evaluate the anti-arthritic and anti-osteoporotic effects of TBD, we conducted H&E straining and safranine O/fast green, TEM, immunohistochemistry (IHC), bone histomorphometry, micro-CT imaging, and biomechanical testing in collagen induced arthritis (CIA) rats. The active ingredient in TBD was identified using network pharmacology and molecular docking. The identification was supported by in vivo IHC assay, and further confirmed using qRT-PCR, Western blot, and SEM analysis in TNF-α-treated MH7A cells and/or in LPS-exposed RAW264.7 cells. RESULTS: Oral administration of TBD attenuated the severity of arthritis and osteopenia as well as poor bone quality, in CIA rats. Additionally, TBD and the positive control, tripterygium glycosides (TG), exhibited similar effects in reducing inflammation in both the synovium and ankle joint. They also were both effective in improving bone loss, microarchitecture, and overall bone quality. TBD reduced the expression of MMP13, IL-17, and p-JNK protein in the synovium of CIA rats. ICA, which was screened, suppressed TNF-α or LPS-triggered inflammatory responses via down-regulating IL-17 signaling, involving in MMP13, IL-1ß, IL-23, and IL-17, and the MAPK pathway including p-ERK, p-JNK, and p-P38, both in MH7A cells and in RAW264.7 cells. Furthermore, ICA prevented osteoclasts from differentiating and bone resoprtion in a dose-dependent manner in vitro. CONCLUSION: This study provides the first evidence that TBD exerts intervening effects on RA-induced OP, possibly through the downregulation of the IL-17/MAPK signaling pathway by ICA. The findings of our study provides valuable insights for further research in this area.

Salvianolate Ameliorates Osteopenia and Improves Bone Quality in Prednisone-Treated Rheumatoid Arthritis Rats by Regulating RANKL/RANK/OPG Signaling.

Rheumatoid arthritis (RA) is closely associated with periarticular osteopenia and leads to a high risk of generalized osteoporosis. Although glucocorticoid (GC) treatment ameliorates joint degradation and manages inflammation in RA, GC application may induce further bone quality deterioration in RA patients. Current treatments for RA lack relevant strategies for the prevention and treatment of osteopenia in RA. In this study, we aimed to investigate whether salvianolate treatment ameliorated osteopenia in prednisone-treated RA rats. Lewis rats with collagen-induced arthritis (CIA) were administered prednisone (PDN) or PDN plus salvianolate (PDN+Sal) treatment for 90 days. The effects of Sal were investigated in PDN-treated CIA rats. To further evaluate the effects of Sal under inflammatory conditions, we investigated the effects of Sal treatment on the TNF-α-induced inflammatory response in MC3T3-E1 osteoblasts. Bone histomorphometry, bone mineral density (BMD), bone biomechanical properties, micro-computed tomography (micro-CT), immunohistochemistry, RT-PCR and western blot analyses were performed to evaluate the effects of Sal. The results demonstrated that RA induced bone loss and bone quality deterioration, with high bone turnover in CIA rats. PDN+Sal treatment significantly increased BMD and trabecular/cortical bone mass, suppressed inflammation, and improved bone biomechanical properties compared to CIA control and PDN treatment. PDN+Sal treatment significantly suppressed bone resorption and the RANKL and RANKL/OPG ratios compared to PDN. PDN+Sal and PDN treatment significantly inhibited TNF-α by 82 and 83%, respectively, and both suppressed inflammation in CIA rats. However, there was no significant difference between PDN+Sal and PDN treatment alone in regard to bone formation parameters or the management of inflammation and arthropathy. Sal significantly increased Osterix, OPN, and Col1a1 while decreasing RANKL, TRAF6, and TRAIL gene in TNF-α-induced MC3T3-E1 osteoblasts. Sal significantly increased Osterix, OPN and RUNX2 while decreasing NF-κB, TRAF6 and IL-1ß protein in TNF-α-induced MC3T3-E1 osteoblasts. The results suggested that salvianolate treatment ameliorated osteopenia and improved bone quality in prednisone-treated RA rats, and the potential mechanism may be related to the regulation of the RANKL/RANK/OPG signaling pathway, TRAIL-TRAF6-NFκB signal axis, and downregulation of inflammatory cytokines. Salvianolate could be used as a promising supplemental therapeutic strategy to ameliorate osteopenia and improve bone quality in GC-treated RA patients.

Exostosin1 as a novel prognostic and predictive biomarker for squamous cell lung carcinoma: A study based on bioinformatics analysis.

The exostosin (EXT) protein family is involved in diverse human diseases. However, the expression and prognostic value of EXT genes in human lung squamous cell carcinoma (LUSC) is not well understood. In this study, we analyzed the association between expression of EXT1 and EXT2 genes and survival in patients with LUSC using bioinformatics resources such as Oncomine and The Cancer Genome Atlas (TCGA) databases, the Gene Expression Profiling Interactive Analysis (GEPIA) server and Kaplan-Meier plotter. Furthermore, regulatory microRNAs (miRNAs) were predicted for EXT1 and used to establish a potential miRNA-messenger RNA (mRNA) regulation network for LUSC using the ENCORI platform. We observed that EXT1 and EXT2 expression levels were higher in LUSC than those in normal tissues. However, only EXT1 expression was significantly associated with poor overall survival (OS) in LUSC patients. Functional annotation enrichment analysis showed that genes co-expressed with the EXT1 gene were enriched in biological processes such as cell adhesion and migration, and KEGG pathways such as extracellular matrix receptor interactions, complement and coagulation cascades, and cell death. Furthermore, three miRNAs, hsa-mir-190a-5p, hsa-mir-195-5p, and hsa-mir-490-3p, were identified to be potentially involved in the regulation of EXT1. In summary, we identified EXT1 expression as a novel potential prognostic marker for human LUSC and the regulatory miRNAs that could possibly contribute to the prognosis of the disease.

Noncoding RNAs: New regulatory code in chondrocyte apoptosis and autophagy.

Osteoarthritis (OA) is a bone and joint disease characterized by progressive cartilage degradation. In the face of global trends of population aging, OA is expected to become the fourth most common disabling disease by 2020. Nevertheless, the detailed pathogenesis of OA has not yet been elucidated. Noncoding RNAs (ncRNAs), including long noncoding RNAs, microRNAs, and circular RNAs, do not encode proteins but have recently emerged as important regulators of apoptosis and autophagy of chondrocytes, thereby highlighting a potential role in chondrocyte injury leading to OA onset and progression. We here review recent findings on these regulatory roles of ncRNAs to provide new directions for research on the pathogenesis of OA and offer new therapeutic targets for prevention and treatment. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.

Ginsenoside Rb1 does not halt osteoporotic bone loss in ovariectomized rats.

Osteoporosis (OP) is a systemic skeletal disorder, manifesting with a reduction in bone mass and deterioration of the microarchitecture. Mesenchymal stem cells (MSCs) have an innate ability to differentiate into several cell types, including osteoblasts (OB). Ginsenoside Rb1 (GRb1) is an ethanol extract from ginseng and contains a highly concentrated form of ginsenoside. GRb1 shows extensive beneficial health effects such as anti-oxidative and anti-inflammatory functions, modulating the immune system and inhibiting osteoclastogenesis. We hypothesized that GRb1 can promote MSC differentiation into OBs and inhibit bone loss. In the present study, we aimed to address two questions: (1) Will GRb1 have a positive effect on osteogenic differentiation of MSCs? and (2) Will GRb1 halt bone loss in ovariectomized (OVX) rats? We investigated the effects of GRb1 on viability and osteogenic differentiation of rat mesenchymal stem cells (rMSCs). Our results showed that GRb1 at concentrations of 10-8 M and 10-6 M can increase alkaline phosphatase activity, mineralization and the expression of osteogenic related proteins, such as osteopontin and osteoprotegerin, while incubating rMSCs with osteogenic induction medium and GRb1. Adding GRb1 into the medium can prevent rMSCs from Oxidative damage at the concentration of 25µM H2O2. Furthermore, 40 4-month-old rats were assigned to 5 groups(8 rats per group): the basal group, the sham group, the OVX group, the high dose of GRb1 group (6 mg/kg/day) and the low dose of GRb1 group (3 mg/kg/day). Rats recrived treatment 3days after surgery and last for 14 weeks. Examinations included serum analysis, mechanical testing, Masson-Goldner trichrome staining and bone histomorphometry analysis. The results showed that OVX can lead to dyslipidemia and excessive oxidative stress, whereas GRb1 cannot significantly halt dyslipidemia and excessive oxidative stress in OVX rats. In addition, the bone density of the lumbar vertebra and femur were decreased significantly in the OVX rats, and GRb1 could not inhibit bone loss. Bone histomorphometry analysis showed that the number and width of bone trabecula of the tibia were reduced in OVX rats, and GRb1 could not prevent their occurrence. A bone biomechanics assay showed that GRb1 cannot improve the ability of bone structure to resist fracture of the femur in OVX rats. The current study demonstrated that GRb1 has an obvious effect on osteogenic differentiation in rMSCs but no obvious effect on bone loss in OVX rats. These findings indicate GRb1 has a positive effect on rMSCs but does not have an effect on bone loss in OVX rats at the concentration we used.

Effect of prednisone treatment for 30 and 90 days on bone metabolism in collagen-induced arthritis (CIA) rats.

Glucocorticoids (GCs) are often prescribed to treat rheumatoid arthritis (RA) in the long term, but there is still controversy in the administration of GCs, mainly because of the adverse reactions such as osteoporosis. Numerous studies have demonstrated that osteoporosis could be induced by GCs in normal rats. However, few experiments have focused on whether osteoporosis could be induced or aggravated by GCs in collagen induced arthritis (CIA) rats. We have investigated bone changes in CIA rats treated with prednisone at 4.5 mg/kg/day for 30 and 90 days by bone histomorphometry, bone mineral density (BMD), micro-CT, biomechanical test, and enzyme-linked immunosorbant assay. We found that high bone turnover osteoporosis was shown in CIA rats. Prednisone treatment for 30 and 90 days improved articular structure and decelerated the degeneration of the femur in CIA rats, but did not improve BMD and bone biomechanics. We conclude that osteoporosis was not aggravated in CIA rats treated with prednisone for 30 and 90 days. On the contrary, prednisone treatment for 30 and 90 days could prevent bone loss of the femur in CIA rats. There was a negative effect on bone metabolism in CIA rats treated with prednisone for 90 days.

Salvianolic acid B and danshensu induce osteogenic differentiation of rat bone marrow stromal stem cells by upregulating the nitric oxide pathway.

The aim of the present study was to investigate the effect of salvianolic acid B (Sal B) and danshensu (DSU) on the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) and the mechanisms of the effects. The osteogenic differentiation of MSCs in culture was assessed by measuring alkaline phosphatase (ALP) activity, osteocalcin (OCN) production, nitric oxide (NO) production and the mRNA expression levels of osteoprotegerin (OPG) and its ligand by MSCs. MSCs were successfully induced to differentiate into osteoblasts and adipocytes. Sal B and DSU increased the ALP activity and the production of OCN in the absence of an ossification inducer. The increase in ALP activity was more pronounced when induction was combined with the osteogenic inducer, Sal B, which enhanced the expression of OPG; however, Sal B reduced the expression of receptor activator of nuclear factor-κB ligand (RANKL) by MSCs. Sal B reversed the inhibitory effect of N-nitro L-arginine methylester on the MSCs and increased ALP activity, OCN content and the OPG/RANKL ratio. Based on these results, it was concluded that Sal B increases the osteogenic differentiation of MSCs, most likely by regulating the nitric oxide pathway.

The induction of autophagy against mitochondria-mediated apoptosis in lung cancer cells by a ruthenium (II) imidazole complex.

In the present study, it was found that the ruthenium (II) imidazole complex [Ru(Im)4(dppz)]2+ (Ru1) could induce significant growth inhibition and apoptosis in A549 and NCI-H460 cells. Apart from the induction of apoptosis, it was reported for the first time that Ru1 induced an autophagic response in A549 and NCI-H460 cells as evidenced by the formation of autophagosomes, acidic vesicular organelles (AVOs), and the up-regulation of LC3-II. Furthermore, scavenging of reactive oxygen species (ROS) by antioxidant NAC or Tiron inhibited the release of cytochrome c, caspase-3 activity, and eventually rescued cancer cells from Ru1-mediated apoptosis, suggesting that Ru1 inducing apoptosis was partially caspase 3-dependent by triggering ROS-mediated mitochondrial dysfunction in A549 and NCI-H460 cells. Further study indicated that the extracellular signal-regulated kinase (ERK) signaling pathway was involved in Ru1-induced autophagy in A549 and NCI-H460 cells. Moreover, blocking autophagy using pharmacological inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) enhanced Ru1-induced apoptosis, indicating the cytoprotective role of autophagy in Ru1-treated A549 and NCI-H460 cells. Finally, the in vivo mice bearing A549 xenografts, Ru1 dosed at 10 or 20 mg/kg significantly inhibited tumor growth.

Effects of salvianolate on bone metabolism in glucocorticoid-treated lupus-prone B6.MRL-Fas (lpr) /J mice.

AIM: To investigate the bone-protective effects of salvianolate (Sal), a total polyphenol from Radix Salviae miltiorrhizae, on bone tissue in the spontaneous lupus-prone mouse model, B6.MRL-Fas (lpr) /J, undergoing glucocorticoid (GC) treatment. METHODS: Fifteen-week-old female B6.MRL-Fas (lpr) /J mice were administered either a daily dose of saline (lupus group), prednisone 6 mg/kg (GC group), Sal 60 mg/kg (Sal group); or GC plus Sal (GC + Sal group) for a duration of 12 weeks. Age-matched female C57BL/6J wild-type (WT) mice were used for control. Micro-computed tomography assessments, bone histomorphometry analysis, bone biomechanical test, immunohistochemistry and immunoblotting analysis for bone markers, and renal histology analysis were performed to support our research endeavor. RESULTS: Lupus mice developed a marked bone loss and deterioration of mechanical properties of bone due to an increase in bone resorption rather than suppression of bone formation. GC treatment strongly inhibited bone formation in lupus mice. Sal treatment significantly attenuated osteogenic inhibition, and also suppressed hyperactive bone resorption, which recovered the bone mass and mechanical properties of bone in both the untreated and GC-treated lupus mice. CONCLUSION: The data support further preclinical investigation of Sal as a potential therapeutic strategy for the treatment of systemic lupus erythematosus-related bone loss.

Secondary osteoporosis in collagen-induced arthritis rats.

Numerous studies have demonstrated that rheumatoid arthritis (RA) is often associated with bone loss; however, few experiments have focused on cancellous and cortical bone changes in rats during the process of arthritis. We have investigated bone changes in rats with collagen-induced arthritis (CIA) and have explored the characteristics of how RA induces osteoporosis by means of bone histomorphometry, bone biomechanics studies, bone mineral density studies, micro computer tomography, enzyme-linked immunosorbant assay, immunohistochemistry, and Western blot analysis. Bone mineral density of the femur and lumbar vertebrae and biomechanical properties of the femur were decreased in CIA rats. Trabecular bone volume of the tibia and lumbar vertebrae was decreased whereas bone resorption was increased in CIA rats. Bone formation of the tibial shaft in periosteal surfaces was decreased in CIA rats. Furthermore, the trabecular bone loss in CIA rats was severer at 16 weeks than at 8 weeks, as was cortical bone loss. The serum level of tumor necrosis factor α in CIA rats was increased, and the expression of dickkopf 1 and that of receptor activator of nuclear factor κB (RANKL) ligand (RANKL) in the ankle joints were also increased, but the expression of osteoprotegerin (OPG) was decreased. We conclude that CIA rats developed systemic osteoporosis, and that osteoporosis became more serious with CIA development. The mechanism may be related to the increase of bone resorption in cancellous bone cause by upregulation of the expression of DKK-1 and regulation of the RANKL/RANK/OPG signaling pathway, and the decrease of bone formation in cortical bone caused by an increase in the expression of DKK-1.

Autophagy plays a protective role in cell death of osteoblasts exposure to lead chloride.

Lead (Pb) is a toxic heavy metal widespreadly used in industrial field. Prior studies showed that Pb exposure had detrimental effects on osteoblasts. The mechanisms underlying Pb-induced damage are complex. Autophagy can protect cells from various cytotoxic stimuli. In the present study, the aim of our research was to investigate whether Pb could activate autophagy to play a protective role against osteoblasts apoptosis. Our results indicated that PbCl2 induced autophagy and autophagic flux in MC3T3-E1 murine osteoblastic cell by RT-PCR, western blot, as well as fluorescence microscopy analysis of GFP-LC3, AO and MDC staining. Pb increased the apoptosis of osteoblasts, evidenced by western blot and Hoechst 33258 staining assessment. In addition, inhibiting autophagy by 3-MA further increased the osteoblasts apoptosis after Pb exposure, showed by flow cytometry and Hoechst 33258 staining. Furthermore, phosphorylation of mTOR and p70S6K was inhibited by Pb exposure, indicating that Pb might induce autophagy in osteoblasts via inhibiting mTOR pathway. Altogether, these evidence suggested that Pb exporsure promoted autophagy flux in osteoblasts. The activation of autophagy by Pb played a protective role in osteoblasts apoptosis, which might be mediated through the mTOR pathway.

Salvianolic acid B prevents bone loss in prednisone-treated rats through stimulation of osteogenesis and bone marrow angiogenesis.

Glucocorticoid (GC) induced osteoporosis (GIO) is caused by the long-term use of GC for treatment of autoimmune and inflammatory diseases. The GC related disruption of bone marrow microcirculation and increased adipogenesis contribute to GIO development. However, neither currently available anti-osteoporosis agent is completely addressed to microcirculation and bone marrow adipogenesis. Salvianolic acid B (Sal B) is a polyphenolic compound from a Chinese herbal medicine, Salvia miltiorrhiza Bunge. The aim of this study was to determine the effects of Sal B on osteoblast bone formation, angiogenesis and adipogenesis-associated GIO by performing marrow adipogenesis and microcirculation dilation and bone histomorphometry analyses. (1) In vivo study: Bone loss in GC treated rats was confirmed by significantly decreased BMD, bone strength, cancellous bone mass and architecture, osteoblast distribution, bone formation, marrow microvessel density and diameter along with down-regulation of marrow BMPs expression and increased adipogenesis. Daily treatment with Sal B (40 mg/kg/d) for 12 weeks in GC male rats prevented GC-induced cancellous bone loss and increased adipogenesis while increasing cancellous bone formation rate with improved local microcirculation by capillary dilation. Treatment with Sal B at a higher dose (80 mg/kg/d) not only prevented GC-induced osteopenia, but also increased cancellous bone mass and thickness, associated with increase of marrow BMPs expression, inhibited adipogenesis and further increased microvessel diameters. (2) In vitro study: In concentration from 10(-6) mol/L to 10(-7) mol/L, Sal B stimulated bone marrow stromal cell (MSC) differentiation to osteoblast and increased osteoblast activities, decreased GC associated adipogenic differentiation by down-regulation of PPARγ mRNA expression, increased Runx2 mRNA expression without osteoblast inducement, and, furthermore, Sal B decreased Dickkopf-1 and increased ß-catenin mRNA expression with or without adipocyte inducement in MSC. We conclude that Sal B prevented bone loss in GC-treated rats through stimulation of osteogenesis, bone marrow angiogenesis and inhibition of adipogenesis.

Preventive effects of berberine on glucocorticoid-induced osteoporosis in rats.

Glucocorticoids are widely used to treat chronic diseases such as rheumatoid arthritis and asthma. However, long-term glucocorticoid therapy can result in serious side effects, such as osteoporosis. The present study investigated the preventive effects of berberine on glucocorticoid-induced osteoporosis in rats. Male Sprague Dawley rats were treated with vehicle, glucocorticoid, glucocorticoid and berberine, or glucocorticoid and calcium carbonate with vitamin D (3) for 12 weeks. The proximal tibiae of all rats were processed without decalcification for quantitative bone histomorphometry, and femur mechanical testing as well as bone mineral density (BMD) were analyzed. A significant decrease was found in the glucocorticoid-treated group compared with the control group in such indices as biomechanical quality, BMD, trabecular bone volume/total tissue area (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), mineral apposition rate (MAR), bone formation rate/total tissue area (BFR/TV), and bone formation rate/trabecular bone surface (BFR/BS). In addition, significantly increased trabecular separation (Tb.Sp), osteoclast number/trabecular bone volume (Oc.N/BV), and osteoclast surface/trabecular bone surface (Oc.S/BS) were observed in the glucocorticoid-treated group, compared with the control group. Berberine and calcium carbonate with vitamin D (3) prevented the decrease in biomechanical quality, BMD, BV/TV, Tb.N, Tb.Th, MAR, BFR/TV, and BFR/BS, as well as increased Tb.Sp, Oc.N/BV, and Oc.S/BS in glucocorticoid-induced osteoporotic rats. The present results suggest that berberine prevents glucocorticoid-induced osteoporosis by inhibiting bone resorption and improving bone formation.

[Effects of DanShenGuBao on biomechanical properties and bone mineral density of femur induced by retinoic acid in rats].

This investigation was directed to the effects of DanShenGuBao on biomechanical properties and bone mineral density (BMD) of femur induced by retinoic acid (RA) in rats. Forty 4-month-old virgin female Sprague-Dawley rats were randomly divided into 5 groups(8 rats each) control group, RA group and different doses of DanShenGuBao groups. Rats in control group were given vehicle, rats in other four groups were given RA at 70 mg x kg(-1) x d(-1) in the morning and given different drugs in the afternoon at the same time. Rats in RA group were given vehicle, rats in other groups were given different doses of DanShenGuBao which contained 10 mg x kg(-1) x d(-1), 5 mg x kg(-1) x d(-1), 2.5 mg x kg(-1) x d(-1) tanshinol, respectively. All of rats were treated at 5 ml x kg(-1) by oral gavaged for 28 days. In preparation for the determination of dynamic changes in bone tissues, all rats were given subcutaneous injections of 30 mg x kg(-1) tetracycline on the 14th, 13th day and 5 mg x kg(-1) calcein on the 4th, 3rd day before death. At the experimental endpoint, the rats were sacrificed by cardiac puncture under sodium pentobarbital anesthesia. Physical parameters, BMD and biomechanical properties of femur were assessed. Compared with those in control group, the physical parameters (cross-sectional diameter, wet and dry weight), BMD and biomechanical properties (max-load, elasticity-load, break-strain, rigid coefficient and bending-energe) were significantly decreased in RA group. Compared with that in RA group, the BMD of femur was increased significantly in medium and high dose of DanShenGuBao group, but there was no significant change in physical parameters and biomechanical properties of femur. RA could decrease the physical parameters, BMD and biomechanical properties of femur. DanShenGuBao could increase BMD, but it was found with no obvious effect on physical parameters and biomechanical properties.

[Correlative changes of histomorphometry and biomechanical property of lumbar vertebrae body in ovariectomized rats].

This paper presents the correlative changes of cancellous bone histomorphometry, biomechanical property and bone mineral contents of lumbar vertebrae in ovariectomized rats. Forty eight 10.5-month-old female SD rats were randomized to six groups: 1) Basal: at 10.5 mon. of age; 2) sham-1: sham-operated at 13 mon. of age; 3) OVX-1: ovariectomized at 13 mon. of age; 4) sham-2: sham-operated at 16 mon. of age; 5) OVX-2: ovariectomized at 16 mon. of age; and 6) OVX-2 + EE: ovariectomized and treated with 17 alpha-ethynylestradiol (100 micrograms.kg-1.d-1) starting at 13 mon. of age for 12 weeks. Double in vivo fluorochrome labeling was administered to all rats. At the end-point of study, the undecalcified longitudinal fourth lumbar vertebra (LV4) sections were cut and stained with Goldner's Trichrome for bone histomorphometric analyses. The mechanical properties of the compact fifth lumbar vertebra body (LV5) were measured with compression test, the LV5 bodies then were dried constantly with high temperature and digested with acid for testing of bone mineral content. A trend in trabecular bone volume (BV/TV), trabecular thickness (Tb.Th) of LV4, bone break load, break stress, elastic moduli and bone calcium content of LV5 to increase with the increase of age was revealed in the intact rats, but the highest value was at 13 mon. of age. A positive correlation was seen between histomophormetry and bone bio-mechanical properties in the intact rats. Compared with the data from the sham-operated rats, the BV/TV and Tb. Th significantly decreased and bone turnover rate significantly increased in OVX rats at 10 weeks, and the above parameters further decreased 22 weeks post OVX. Correspondingly, the bone break load, break stress, elastic moduli and bone calcium content significantly decreased with the increase of time post OVX was compared with those of sham-operated rats. A positive correlation between histomophormetry and bone biomechanical properties was also observed in OVX rats (r = 0.399, P = 0.018). Bone histomorphometry is good for predicting early physiological and pathological changes and bone biomechanical properties are good parameters for predicting corresponding function of bone such as fracture. Both of them are useful and needful in the animal studies on osteoporosis and related pharmacodynamic observations on new drugs.

[Synthetical evaluation of promoting effect of some kinds of transdermal enhancers with grey relational cluster method].

OBJECTIVE: Synthetical evaluation of promoting effect of some kinds of transdermal enhancers was carried through. METHOD: Diclofenac sodium was used as model, and azone and l-menthol and synthetic borneol and olieic acid and essential oil from Cnidium monnieri were used as transdermal enhancers. Transdermal absorption experimentation of diclofenac sodium on the device of penetrating skins in vitro was done. Cumulation of permeation amount and penetrating rates and steady fluxes and lag times were observed, and grey relational cluster method was used to evaluate the promoting effect of some kinds of transdermal enhancers. RESULT: As for promoting effect on diclofenac sodium, azone and l-menthol were the best, and synthetic borneol and olieic acid ranked behind. CONCLUSION: Grey relational cluster method can evaluate promoting effect objectively and fairly.