Molecular mechanism of Chinese medicine formula (Bushen Bugu Tang) for the treatment of postmenopausal osteoporosis

AIM: To study the molecular mechanism of Chinese medicine formula Bushen Bugu Tang (BSBGT) for the treatment of postmenopausal osteoporosis (PMO). METHODS: Forty female SD rats were divided into four groups: the sham group, OVX group, estrogen treatment group and BSBGT treatment group, ten for each, difference operation and treatment were given. Subsequently, bone mineral density (BMD), bone histomorphometric changes and collagen type I expression in the femur of these rats were examined. In order to position the target of the formula acting on bone, the effect of BSBGT on regulation of mRNA levels of RANKL and OPG, two key factors for regulating osteoclastogenesis, by using rat calvarial-derived stromal cell cultrues, was elucidated. RESULTS: The BSBGT formula was found as effective as estrogen in preventing the reduction of BMD, the trabecular area and trabecular numbers in femora and also the decrease in collagen type I expression following ovariectomy. RT-PCR resulted showed that both estrogen and BSBGT significantly up-regulated OPG mRNA level and down-regulated RANKL mRNA level, resulting in an increased ratio of OPG/RANKL in cultured rat stromal cells. CONCLUSIONS: The ratio of OPG/RANKL within the osseous environment determines whether bone formation predominates bone resorption. We therefore concluded that the BSBGT formula might be as effective as estrogen in prevention of postmenopausal osteoporosis through the mechanism of up-regulating RANKL expression and down-regulating OPG expression in osteoblast/stromal cells.     

Effects of exercise combined with forsamax on bone mineral density and nerve conduction in ovariectomy-induced osteoporosis rats

AIM: To observe the effects of exercise (EX) and forsamax (FOX) on bone mineral density (BMD) and nerve conduction in ovariectomized rats, and to determine whether exercise can enhance the effects of forsamax on postmenopausal osteoporosis (PMOP). METHODS: Ninety 6-month-old female SD rats were randomly divided into 2 groups: sham group (18 rats) and ovariectomized group (72 rats). Eight weeks after ovariectomy, dual-energy X-ray absorptiometry was applied to scan the BMD of the fourth lumbar vertebra. The level of serum estradiol (E₂) was analyzed by ELISA. The living ovariectomized rats were then divided into 4 groups: ovariectomized model group (OVX), fosamax treatment group (OVX+FOX), exercise treatment group (OVX+EX), and exercise combined with fosamax treatment group (OVX+FOX+EX). After treated with fosamax (1 mg/kg intragastrically) and/or exercise for 12 weeks, the BMD of the fourth lumbar vertebra, the motor nerve conduction velocity (MCV), motor distal latency (ML) and compound muscle action potential (CMAP) of the left femoral nerve were detected. The levels of serum type 1 procollagen carboxy-terminal propeptide (PICP) and type I collagen carboxy-terminal cross-linked telopeptide (ICTP) were also analyzed by ELISA. RESULTS: Twelve weeks after FOX and/or EX treatment, ovariectomized rats showed obviously lower BMD, higher PICP and ICTP than those in sham group (P<0.05). FOX or EX significantly increased BMD and reduced PICP induced by ovariectomy (P<0.05). FOX significantly reduced ICTP (P<0.05), but no remarkable difference was observed in OVX+FOX group as compared with OVX group. EX+FOX significantly increased BMD and reduced PICP and ICTP compared with FOX or EX alone (P<0.05). However, no obvious difference was observed between OVX+FOX group and OVX+EX group. No distinct difference in MCV and CMAP among the 5 groups was found. Neither ovariectomy nor FOX significantly affected ML. EX or EX+FOX made ML shorter than that in OVX group (fP<0.05), and ML was remarkably shortened in OVX+FOX+EX group than that in FOX alone group on the left femoral nerve (P<0.05). No significant difference between FOX and EX in the protective effects against ovariectomized rats was observed. CONCLUSION: Exercise and fosamax may restrain bone absorption effect of osteoclast and then improve the bone mineral density in ovariectomized rats.     

Relationship between changes of bone mineral density and bone marrow angiogenesis in ovariectomized rats

AIM: To explore the relationship between the changes of bone mineral density (BMD) and bone marrow angiogenesis in ovariectomized rats. METHODS: Thirty female Sprague-Dawley rats (3 month-age) were randomly divided into ovariectomized (OVX) groups and sham operated (sham) groups, and executed after 4 weeks, 8 weeks and 12 weeks respectively. The bone mineral density (BMD) of left femora was measured. The right distal femoral epiphysis was fixed in formalin, decalcificated by EDTA-Na2, dehydrated and embedded in paraffin. Four-μm-thick slices were obtained from the paraffin section and stained with haematoxylin-eosin (HE) for bone marrow pathological examination. The number of bone marrow microvessels was examined by means of immunohistochemical staining for CD34 to stain the endothelial cells of blood vessels to definite the bone marrow microvascular density (MVD). Plasma levels of vascular endothelial growth factor (VEGF) were examined by the method of ELISA. RESULTS: The BMD of femoral in 8-week OVX group were decreased significantly compared to the 8-week sham group (P<0.05), suggesting the establishment of osteoporosis model. Meanwhile, the area of hematopoietic tissue decreased and the area of adipose tissue increased. These changes became obviously in 12-week OVX group, and the area of trabecular bone and the bone marrow MVD significantly decreased compared to the 12-week sham group. A positive correlation between MVD and BMD, area of hematopoietic or trabecular bone as well as a negative correlation between MVD and area of adipose tissue were observed. The plasma levels of VEGF in OVX group were not significantly different from that in sham group, and had no correlation relationship with the indexes of bone marrow pathology. CONCLUSION: There has an increase in MVD companied with the bone mass loss and hematopoietic tissue decreased in ovarietomized rats, which provide experiment proof to treat osteoporosis with the means of promoting of MVD in bone marrow.     

Histomorphometry investigation and mechanism exploration of postmenopausal osteoporosis

AIM: To explore the pathophysiological mechanism of postmenopausal osteoporosis, we established an animal model for postmenopausal osteoporosis and investigated the loss of bone mass.METHODS: Thirty-one female SD rats (three months old) were randomly divided into two groups: sixteen ovariectomized (OVX) and fifteen sham-operated group as control. At post-operated day 28, seven rats of either OVX group or sham-operated group were sacrificed. At post-operated day 56, the rest of either were sacrificed. The wet weight of the uteruses were measured and the changes of bone micro-architecture were studied by bone mineral density (BMD) and the data of bone histomorphometry. RESULTS: Compared with the sham group, the mean wet weight and the bone density of proximal tibia 1/3 section of the OVX group was significantly decreased (P＜0.05); Data of bone histomorphormetry showed that the trabecular percentage area of metaphysis of both distal femur and proximal tibia was significantly decreased (P＜0.01) both at post-operated day 28 and day 56, so were the number of trabecular bone and the thickness of trabecular bone; but no significance was observed in epiphysis.CONCLUSION: BMD and the data of bone histomorphometry at distal femur decreased steadily while those of at proximal tibia did sharply and unsteadily; the early loss of bone mass after ovariectomy was found in metaphysis of long bones.     

Rosiglitazone inhibits osteoclastogenesis in rheumatoid arthritis by down-regulating RANKL expression and suppressing ERK phosphorylation

AIM: To investigate the effects of rosiglitazone on fibroblast-like synoviocyte (FLS)-induced osteoclastogenesis in rheumatoid arthritis (RA) and the related mechanism. METHODS: RA-FLS were cocultured with peripheral blood monocytes from healthy volunteers in the presence of macrophage colony-stimulating factor (M-CSF) and rosiglitazone. Osteoclasts were assayed by tartrate-resistant acid phosphatase (TRAP) staining. Resorption lacunae area was identified by toluidine blue staining and quantified by image analysis software. The mRNA expression of RANKL and OPG was evaluated by real-time PCR, and the protein levels of RANKL, OPG, p-ERK, p-p38 and p-JNK were measured by Western blot. RESULTS: Compared with control group (without rosiglitazone treatment), rosiglitazone at concentration of 15 μmol/L significantly decreased the number of osteoclasts (P<0.01) and resorption lacunae area (P<0.05). The expression of RANKL at mRNA and protein levels was significantly down-regulated by rosiglitazone at concentration of 15 μmol/L, while the mRNA and protein expression of OPG was up-regulated (P<0.01). Rosiglitazone (15 μmol/L) significantly decreased the protein level of p-ERK (P<0.05), but not the protein level of p-p38 or p-JNK (P>0.05). CONCLUSION: Rosiglitazone inhibits RA-FLS-induced osteoclast formation and its resorption activity by down-regulating RANKL expression and ERK phosphorylation, suggesting that rosiglitazone may inhibit RA osteoclastogenesis and bone resorption.     

“中国园艺学会十届二次理事会暨学术研讨会”预备通知

AIM:The aim of the present study is to investigate changes of five biochemical markers of bone turnover during the formation of ovariectomized rat model. METHODS:Three-month-old female SD rats were divided randomly into ovariectomized (OVX) group, sham-operated (sham) group and control group. Five biochemical markers of bone turnover levels, including serum osteocalcin (OC), total alkaline phosphatase (ALP), bone-specific alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP) and hydroxyproline (HYP) were measured before and at 1, 1.5, 2, 2.5, 3 and 4 months after surgery. The right proximal tibias of rats were excised at the same time point for histopathological observation. RESULTS:Serum OC, ALP, BALP, TRAP and HYP levels in OVX group were significantly higher than that in sham group. As regard to the time of peak level arrived, the sequence of changes was as follows: TRAP/HYP→OC→ALP/BALP. There was a significant positive correlation between five markers. The pathological changes of trabecular bone in OVX group were only observed 3 months after surgery. CONCLUSION:The results suggest that postmenopausal osteoporosis has a high bone turnover rate. During the formation of ovariectomized rat model, changes of bone resorption markers precede changes of bone formation markers. Serum OC, ALP, BALP, TRAP and HYP are sensitive to evaluate the bone loss in the earlier stage of postmenopausal osteoporosis.     

Effects of drynaria total flavonoids on serum levels of leptin,interleukin 6, prostaglandin E2 and expression of bone β2-adrenergic receptor in ovariectomized osteoporosis rats

AIM: To investigate the effects of drynaria total flavonoids on serum levels of leptin (LEP), interleukin 6(IL-6), prostaglandin E₂(PGE₂) and the expression of bone β₂-adrenergic receptor (ADRB2) in a rat model of ovariectomized osteoporosis(OP). METHODS: The osteoporosis model was established by ovariectomy. Twelve weeks after modeling,bone mineral density (BMD) was determined by dual-energy X-ray absorptiometry to verify successful modeling.Enzyme-linked immunosorbent assay was applied to detect the concentrations of LEP, IL-6 and PGE₂ in serum. The expression of ADRB2 was determined by immunohistochemical technique. RESULTS: Compared with sham group,BMD of the rats in model group significantly decreased in multiple regions 12 weeks after modeling(P<0.01). The serum levels of LEP, IL-6 and PGE₂ in model group were significantly higher than those in sham group(P<0.05). The levels of LEP, IL-6 and PGE₂ in drynaria total flavonoids group were significantly lower than those in model group(P<0.01). No significant difference of PGE₂ between these 2 groups was observed. The ADRB2 expression in sham group and treatment group was significantly different from that in model group, and no significant difference between sham group and treatment group was found. CONCLUSION: The serum levels of LEP, IL-6 and PGE₂ and the expression of bone ADRB2 increased in OP rats.Drynaria total flavonoids reduce the production of LEP, IL-6 and the expression of ADRB2, and suppress the bone absorption, which may be one of the mechanisms in treating OP.     

Effects of folic acid on antioxidant enzyme,nitric oxide synthase and nitric oxide in ovariectomized rats

AIM: To observe the effects of folic acid (FA) on antioxidant enzyme, nitric oxide synthase (NOS) and nitric oxide (NO) in ovariectomized (OVX) rats.METHODS: Forty three-month-old female SD rats were randomly divided into 5 groups: sham group, OVX group, diethylstilbestrol group (0.03 mg·kg^-1·d^-1), low-dose FA group (5 mg·kg^-1·d^-1) and high-dose FA group (20 mg·kg^-1·d^-1). Gastric gavage started 1 week after operation and lasted for 10 weeks. The rats in sham group and OVX group were given distilled water instead of FA as controls. At the end of the 10th week, the L₅ vertebra and right femur were removed for determination of bone mineral density (BMD). The bone homogenates were made using the L₃ and L₄ vertebrae. The levels of the total antioxidant capacity (TAC), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), NOS and NO were detected in plasma and bone homogenates.RESULTS: Compared with sham group, the BMD levels in L₅ vertebra and right femur and the levels of GSH-Px and NO in the plasma were all decreased. The levels of TAC, GSH-Px, NOS and NO in the bone homogenates were also decreased, while the MDA concentration was increased in OVX group (all P < 0.01). Compared with OVX group, the levels of TAC, GSH-Px, NOS, NO and BMD of the L₅ vertebra and right femur were all increased, while the MDA concentration was decreased in high-dose FA group (all P < 0.01). CONCLUSION: In female SD rats, ovariectomy leads to a significant reduction of antioxidant enzyme, NOS and NO levels. Oxidative stress is possibly involved in the development of osteoporosis. Protection against osteoporosis by high-dose FA may be linked to improvement of antioxidant enzyme activity, the levels of NOS and NO as well as a reduction of oxidative stress in ovariectomized rats.     

The relationship between serum IGF-1, IGFBP-3 levels,bone mineral density and bone metabolic markers in postmenopausal patients with osteoporosis

AIM:To study the relationship between serum insulin-like growth factor- 1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3) levels, bone mineral density(BMD) and bone metabolic markers in postmenopausal women. METHODS:Serum IGF-1, IGFBP-3, osteocalcin(BGP), isomeric C-telopeptide of type I collagen (β-CTX), estradiol(E₂), calcitonin(CT), parathormone(PTH), calcium (Ca), and phosphorus(P) were measured in 90 postmenopausal osteoporosis patients and 70 healthy postmenopausal women. BMD of lumbar vertebra and left femoral neck were determined by dual energy X-ray absorptiometry.RESULTS:BMD of lumbar vertebra and left femoral neck decreased significantly (P<0.01), serum IGF-1, IGFBP-3, E₂, CT and BGP decreased significantly (P<0.01), serum β-CTX and PTH increased significantly in postmenopausal osteoporosis group (P<0.01). There were no significantly differences in serum Ca, P between two groups (P>0.05). BMD of lumbar vertebra and left femoral neck were positively correlated with serum IGF-1, IGFBP-3, E₂, CT and BGP, but negatively correlated with β CTX and PTH. There were no correlation with serum Ca, P and BMD.CONCLUSION:Serum IGF-1, IGFBP-3, E₂, CT, BGP, β-CTX and PTH level were correlated with BMD of lumbar vertebra and left femoral neck. These markers can be one of the valuable evidences for screening osteoporosis in postmenopausal women.     

Effect of 17β-estradiol on the levels of plasma oxidized low density lipoprotein in ovariectomized rabbits

AIM: To observe the changes of plasma oxidized low density lipoprotein(oxLDL) levels of ovariectomized cholesterol-fed rabbits with or without 17β-estradiol(E₂) replacement therapy.METHODS: All rabbits were ovariectomized and fed standard chow supplemented with 1.5% cholesterol for 14 weeks. Two weeks after operation, the rabbits were randomly assigned to four groups. Three groups were treated with E₂ 1 mg, 2 mg, 4 mg respectively, the other group served as control. Serum superoxide dismutase (SOD) levels and plasma oxLDL levels were measured at 0, 3, 8, 12 weeks after hormone replacement therapy. The aortic atherosclerotic lesion areas were determined by computer. RESULTS: We found that there were striking increase of serum SOD levels ( P<0.05 ) and significant decrease in both the plasma oxLDL levels and the aortic atherosclerotic lesion areas ( P<0.01 respectively). Moreover, a positive correlation was found between plasma oxLDL levels and the areas of atherosclerotic plaque in all rabbits. CONCLUSIONS: Estrogen attenuates atherogenesis in cholesterol-fed ovariectomized rabbits. And this beneficial effect of E₂ may be duo to its lowering of plasma oxLDL level.     

Effects of Yigu capsule on the level of cbf α-1 mRNA in bone of ovariectomized osteoporosis rats

AIM: To analyze the regulatory effect of Yigu capsule on core binding factor alpha 1 (cbf α-1) gene expression in bone of ovariectomized osteoporosis (OP) rats. METHODS: Thirty-six 10-month old Sprague-Dawley female rats were randomized into three groups: sham-operated group, model group and drug group. After intervention by the corresponding methods, the femurs were collected, SYBR green Ⅰ fluorescence quantitative PCR technique was applied with the internal control of GAPDH according to the relative quantitative formula (2-ΔΔCt), the differentially expressed multiples between the model group or drug group and sham-operated group were calculated. RESULTS: Quantitative formula analysis showed that the level of cbf α-1 gene expression in bone of model groups was decreased than that in sham-operated rats ［compared with sham-operated group, P<0.01, it was (9.9×105)-(1.6×104) times］. While in drug groups the level of cbf α-1 gene expression was 0.19 to 0.92 times than that in the sham-operated group, no significant difference was observed (P>0.05). CONCLUSION: The results of this study show that cbf α-1 gene expression in bone tissue of ovariectomized OP rat is decreased, and Yigu capsule increases the level of cbf α-1 gene expression in bone of OP, indicating that Yigu capsule induces bone marrow mesenchymal stem cells into osteoblasts.     

Influence of ginsenoside Re on vascular intima hyperplasia and NF-κB p65 signaling pathway in balloon-injured rats

AIM: To investigate the inhibitory effect of ginsenoside Re on intimal hyperplasia induced by balloon-injury and to explore the role of NF-κB p65 signaling pathway in the process. METHODS: SD rats(n=40) were divided into 5 groups randomly: sham operation group, model group, low-dose ginsenoside Re group, middle-dose ginsenoside Re group and high-dose ginsenoside Re group. The carotid artery intima injury model was established by 2F balloon catheters in all groups except the sham operation group. The day after modeling, the animals in model group and sham operation group were administered intragastrically with distilled water, and the rats in low-dose, middle-dose and high-dose ginsenoside Re groups were given ginsenoside Re at doses of 12.5 mg/kg, 25mg/kg and 50 mg/kg, respectively. After 14 continuous days, the morphological changes of the injured arteries were observed by HE staining and the lumen area, intima area and media area as well as the ratio of intimal area/media area were determined. The expression of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) were detected by real-time PCR. The proliferating cell nuclear antigen(PCNA) and nuclear factor-kappa B(NF-κB) p65 were examined by immunohistochemistry.RESULTS: Compared with sham operation group, the vessel cavity was narrowed(P<0.01), the mRNA levels of TNF-α and IL-1β, and the protein expression of PCNA and NF-κB p65 were increased in model group(P<0.05). Compared with model group, the vascular intimal hyperplasia was alleviated obviously(P<0.05), and the mRNA levels of TNF-α and IL-1β, and protein expression of PCNA and NF-κB p65 were decreased in medium and high-dose ginsenoside Re groups(P<0.05). CONCLUSION: Ginsenoside Re inhibits the vascular neointimal hyperplasia induced by balloon-injury in rats, and the molecular mechanism may be related to the inhibition of NF-κB p65 signaling pathway.     

The roles of ERK signaling pathway in osteogenic differentiation of rat MSCs promoted by quercetin

AIM:To study the roles of extracellular signal-regulated kinase(ERK) signal pathway in the process of osteogenic differentiation in rat mesenchymal stem cells(MSCs) promoted by quercetin(QUE). METHODS:The optimal concentration of QUE for promoting osteogenic differentiation of rat MSCs was determined by MTT and alkaline phosphatase(ALP) detection. The activity of ALP was detected by the ALP detection kit. The expression of bone Gla protein(BGP) and collagen typeⅠ(ColⅠ) was observed by ELISA analysis. MSCs were exposed to QUE at optimal concentration with or without ERK1/2 inhibitor PD98059. Non-phosphorylated and phosphorylated expression of ERK1/2 was analyzed by Western blotting. The mRNA expression of transforming growth factor β₁(TGF-β₁), bone morphogenetic protein 2(BMP-2) and core binding factor α1(Cbfα1) was measured by fluorescence quantitative PCR. RESULTS:QUE at concentrations of 0.1 μmol/L, 1 μmol/L and 10 μmol/L induced the expression of ALP in MSCs in a dose-dependent manner, and also promoted MSCs proliferation. The expression levels of ALP, BGP and ColⅠwere higher in QUE group, and was lower in PD89059 group than those in control group. Compared with control group, the level of phosphorylated ERK1/2, and the mRNA expression of TGF-β₁, BMP-2 and Cbfα1 increased in QUE group. The mRNA expression of TGF-β₁, BMP-2 and Cbfα1 in QUE+PD98059 group decreased as compared with QUE group. CONCLUSION:QUE promotes osteogenic differentiation of MSCs by activating ERK signaling pathway.     

Effect of dexamethasone on differentiation of human osteoblast and osteoclast in vitro

AIM: To investigate the effect of dexamethasone (DEX) on differentiation of human osteoblasts (OBs) and osteoclasts (OCs) in vitro.METHODS: The normal human bone marrow mesenchymal stem cells (MSCs) were stimulated with the inducer mainly containing DEX in vitro. Alizarin red staining and alkaline phosphatase (ALP) kit were used to identify the formation of calcium nodules among OBs and ALP levels in OBs,respectively. The levels of ALP were quantified again after the concentration of DEX was changed, and the expression of phosphorylated GSK-3β in OBs was detected by Western blotting. Human bone marrow mononuclear cells (BMMs) were induced by exogenous receptor activator of nuclear factor kappa B ligand (RANKL) for 14 days. Tartrate-resistant acidic phosphatase staining was used to identity the formation of OCs, and the downstream signaling pathway of RANKL was detected by Western blotting. The le-vel of soluble RANKL(sRANKL) in the supernatants and the expression of RANKL in MSCs were measured by ELISA and Western blotting,respectively,when MSCs were stimulated with different concentrations of DEX for 7 days.RESULTS: Calcium nodules were formed when MSCs were stimulated with DEX at concentration of 0.1 μmol/L. The level of ALP and the protein expression of phospho-GSK-3β in OBs gradually increased by treating the cells with DEX at the concentrations within the range of 0.001-0.1 μmol/L. However, the level of ALP and the protein expression of phospho-GSK-3β were decreased as the concentrations of DEX further increased to the range of 0.1-10 μmol/L. BMMs were differeniated into OCs by exogenous RANKL, and the phosphorylations of IκBα, SPAK/JNK, p38 MAPK and p44/42 MAPK in BMMs increased. When MSCs were stimulated with DEX at the concentrations within 0.001-10 μmol/L, the level of sRANKL in the supernatants and the protein expression of RANKL in MSCs gradually increased, and the level of sRANKL was positively correlated with the concentration of DEX (r=0.821,P<0.05). CONCLUSION: DEX at low concentrations promotes the differentiation of OBs and OCs. DEX at high concentrations only promotes the differentiation of OCs, not that of OBs.     

Akebia saponin D promotes differentiation of bone marrow mesenchymal stem cells into osteoblasts through MAPK signaling pathways

AIM:To explore the role of Akebia saponin D(ASD) in the differentiation of rat bone marrow-derived mesenchymal stem cells(BMSCs) into osteoblasts. METHODS:The rat BMSCs were cultured using routine methods. The effects of ASD on the differentiation of MSCs into osteoblasts were observed. The p38 mitogen-activated protein kinase(p38 MAPK) inhibitor SB203580 and extracellular signal-regulated kinase(ERK) inhibitor PD098059 were used to evaluate the mechanisms. The activity of alkaline phosphate(ALP) and content of osteocalcin(OC) were assayed during differentiation. The mRNA expression of osteoprotegerin(OPG) and receptor activator of nuclear factor-κB ligand(RANKL) was determined by real-time fluorescence quantitative PCR. The activity of p38 MAPK and ERK was measured by Western blotting. RESULTS:Six days after treatment with ASD, the mRNA expression of OPG significantly increased, while the mRNA level of RANKL significantly decreased in induced cells. ASD increased the activity of ALP and the content of OC. Moreover, ASD enhanced the activity of both p38 MAPK and ERK, which was inhibited by SB203580 and PD098059. SB203580 and PD098059 also inhibited the positive role of ASD in the differentiation of MSCs into osteoblasts. CONCLUSION:Akebia saponin D significantly enhances differentiation of rat BMSCs into osteoblasts in vitro, which may be mediated by the p38 MAPK and ERK signaling pathways.     

Effects of naringin on MAPK signal pathway in rat bone marrow mesenchymal stem cells during osteogenic differentiation

AIM: To study the effects of Chinese herbal monomer naringin (NG) on the MAPK signal pathway in bone marrow mesenchymal stem cells (MSCs) derived from SD rats during the differentiation into osteoblasts in vitro . METHODS: The changes of evaluating indicators alkaline phosphatase (ALP), bone gla protein (BGP) and type I collagen (Col I) in MSCs were observed under the conditions of normal, adding p38 pathway inhibitor SB203580, adding extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059, adding c-Jun N-terminal kinase (JNK) pathway inhibitor SP600125, and adding SB203580, PD98059 and SP600125 together. The protein phosphorylation of p38, ERK1/2 and JNK was measured by Western blotting. The mRNA expression levels of transforming growth factor beta 1 (TGF-β₁), bone morphogenetic protein 2 (BMP-2) and core binding factor α1 (Cbfα1) were measured by fluorescence quantitative PCR. RESULTS: The most effective concentration of NG to promote the differentiation of MSCs into osteoblasts was 10^-7 mol/L. The highest expression levels of both ALP and BGP were observed in NG group (P<0.05), while the expression of Col I did not reveal significant difference (P>0.05). Compared with NG group, the expression levels of ALP, BGP and Col I decreased differently after adding different inhibitors. Compared with control group, the protein phosphorylation of JNK was increased (P<0.05), and the phosphorylation of p38 was decreased (P<0.05), while the phosphorylation of ERK1/2 did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the protein phosphorylation of p38, ERK1/2 and JNK showed fluctuation with some increasing and others decreasing. Compared with control group, the expression of BMP-2 was increased (P<0.05), and the expression of Cbfα1 was decreased(P<0.05), while the expression of TGF-β₁ did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the mRNA expression levels of TGF-β₁, BMP-2 and Cbfα1 decreased differently after adding different inhibitors. CONCLUSION: Activation of ERK/JNK signaling and up-regulation of BMP-2 expression may be the main mechanism of NG to promote the differentiation of MSCs into osteoblasts. NG has strong impact on p38 pathway to improve the expression of BMP-2 in MSCs.     

Hypolipemic and hypoglycemic effects of total triterpenoids from Psidium guajava leaves on type 2 diabetic rats

AIM: To investigate the effects of total triterpenoids from Psidium guajava leaves (TTPGL) on blood glucose and lipids in type 2 diabetic rats. METHODS: The diabetic rats were induced by intraperitoneal injection of streptozotocin at dose of 35 mg/kg and feeding with high-fat diet. The animals were divided into 5 groups: diabetic model control group (model), TTPGL treatment groups (with the doses of 60, 120 and 240 mg/kg, respectively) and rosiglitazone treatment group (3 mg/kg). Another 12 normal SD rats were used as the normal controls. The rats received daily treatment for 6 weeks, and then the levels of fasting blood glucose (FBG), fasting insulin (FINS), triglyceride (TG), total cholesterol (TCH), free fatty acid (FFA), glycosylated hemoglobin (GHb) and glycosylated serum proteins (GSP) were measured. The protein expression of peroxisome proliferator-activated receptor γ (PPARγ) in adipose tissues was detected by Western blotting. RESULTS: Compared with normal control group, the levels of FBG, GHb and blood lipids were increased in type 2 diabetic rats. The FINS, insulin sensitivity index, and the protein expression of PPARγ in adipose tissues were decreased. Compared with model group, the levels of FBG and GSP were decreased,and the FINS, insulin sensitivity index, and the protein expression of PPARγ in adipose tissues significantly increased in TTPGL treatment groups (with the doses of 120 and 240 mg/kg). The levels of serum TG,TCH and FFA were significantly lower in TTPGL treatment groups (P<0.01 or P<0.05) as compared with the model controls. CONCLUSION: TTPGL decreases the levels of blood glucose and lipids in diabetic rats. TTPGL also increases serum insulin level and improves insulin sensitivity. The action mechanism of TTPGL may be related to the increase in the protein expression of PPARγ.     

Effects of different lighting on reproductive system in depressive female rats

AIM: To investigate the effects of different lighting on the reproductive system in depressive female rats. METHODS: Healthy adult female rats were randomly chosen as control group, and the depressive adult female rats in SPF grade were randomly divided into 5 groups(7 rats each):depressive model group, sulfur lamp group, heat radiation lamp group, fluorescent lamp group and LED lamp group. After 45 d of continuous illumination, the estrous cycle was observed by the vaginal exfoliated cells, and the organ indexes of ovary and uterus were calculated. The concentrations of estiadrol(E₂), prolactin(PRL), progesterone(PROG) and testosterone(T) in the serum were detected by ELISA, and the histopathological lesion of ovary was observed under microscope with HE staining. RESULTS: The estrous cycle exhibited serious disorder, the ovaries exhibited serious congestion, and the organ indexes of ovary and uterus and the concentrations of E₂, PRL, PROG and T decreased significantly in the rats in depressive model group compared with control group(P<0.05). The estrous cycle and histopathological damage of ovary were obviously improved, and the concentrations of E₂, PRL, PROG and T were significantly increased after the sulfur lamp lighting in the depressive female rats compared with depressive model group. No obvious change and improvement of the reproductive functions in the heat radiation lamp group, fluorescent lamp group and LED lamp group was observed. CONCLUSION: The reproductive functions of depressive female rats are improved by sulfur lamp lighting.     

Role of estrogen on expression of TNF-α and MMP-9 in intracranial arterial vascular wall of spontaneous hypertensive rats

AIM: To study the effects of estrogen on the inflammatory response and vascular remodeling of intracranial artery in rats.METHODS: Thirty-two female spontaneous hypertensive rats (SHR) were randomized into 4 groups: spontaneous hypertensive group(sham-operated), ovariectomized group, ovariectomized+17 beta-estradial group and ovariectomized+vehicle group (8 rats in each group).On day 14, estradiol was detected by radioimmunoassay.The pathological changes were observed under light microscope.The protein expression of tumor necrosis factor alpha (TNF-α) and matrix metalloproteinase-9 (MMP-9) in vascular wall of Willis circle was detected by Western blotting.RESULTS: The estrogen level was lower in ovariectomized group than that in sham-operated group (P<0.01).The estrogen level was higher in ovariectomized rats treated with 17 beta-estradial than that in ovariectomized rats treated with vehicle (P<0.01).Advanced aneurysm was not found in all groups.Early aneurysmal change was not found in sham-operated group.Early aneurysmal changes in some rats were observed in ovariectomized group (2 rats), ovariectomized+vehicle group (3 rats) and ovariectomized+17 beta-estradial group (1 rat).The protein levels of TNF-α and MMP-9 in the vascular wall of Willis circle in sham-operated group were lower than that in ovariectomized group (P<0.01).Additionally, the protein levels of TNF-α and MMP-9 in the vascular wall of Willis circle of ovariectomized rats treated with 17 beta-estradial were lower than those of ovariectomized rats treated with the vehicle (P<0.01).CONCLUSION: Estrogen can influence the vascular remodeling of intracranial artery by inhibiting the inflammatory response and degradating MMP-9 in the vascular wall.