Simulated microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts can be prevented by protection of primary cilia.

Oxidative stress has been considered to be closely related to spaceflight-induced bone loss; however, mechanism is elusive and there are no effective countermeasures. Using cultured rat calvarial osteoblasts exposed to microgravity simulated by a random positioning machine, this study addressed the hypotheses that microgravity-induced shortening of primary cilia leads to oxidative stress and that primary cilium protection prevents oxidative stress and osteogenesis loss. Microgravity was found to induce oxidative stress (as represented by increased levels of reactive oxygen species (ROS) and malondialdehyde production, and decreased activities of antioxidant enzymes), which was perfectly replicated in osteoblasts growing in NG with abrogated primary cilia (created by transfection of an interfering RNA), suggesting the possibility that shortening of primary cilia leads to oxidative stress. Oxidative stress was accompanied by mitochondrial dysfunction (represented by increased mitochondrial ROS and decreased mitochondrial membrane potential) and intracellular Ca2+ overload, and the latter was found to be caused by increased activity of Ca2+ channel transient receptor potential vanilloid 4 (TRPV4), as also evidenced by TRPV4 agonist GSK1016790A-elicited Ca2+ influx. Supplementation of HC-067047, a specific antagonist of TRPV4, attenuated microgravity-induced mitochondrial dysfunction, oxidative stress, and osteogenesis loss. Although TRPV4 was found localized in primary cilia and expressed at low levels in NG, microgravity-induced shortening of primary cilia led to increased TRPV4 levels and Ca2+ influx. When primary cilia were protected by miR-129-3p overexpression or supplementation with a natural flavonoid moslosooflavone, microgravity-induced increased TRPV4 expression, mitochondrial dysfunction, oxidative stress, and osteogenesis loss were all prevented. Our data revealed a new mechanism that primary cilia function as a controller for TRPV4 expression. Microgravity-induced injury on primary cilia leads to increased expression and overactive channel of TRPV4, causing intracellular Ca2+ overload and oxidative stress, and primary cilium protection could be an effective countermeasure against microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts.

Protection of primary cilia is an effective countermeasure against the impairment of osteoblast function induced by simulated microgravity.

The molecular mechanism for the microgravity-induced decrease in bone formation remains unclear and there is a lack of effective specific preventative therapies. We recently reported that primary cilia of osteoblasts became shorter and even disappeared when the cells were exposed to random positioning machine (RPM)-simulated microgravity and that the microgravity-induced loss of osteogenic potential of osteoblasts could be attenuated when the resorption of primary cilia was prevented by treatment with 0.1 µM cytochalasin D. In the current study, it was further found that the loss of the osteogenic capacity of rat calvarial osteoblasts (ROBs) was associated with the inhibition of the BMP-2/Smad1/5/8 signalling pathway, of which most of the signalling proteins including BMP-2, BMPRII, Smad1/5/8 and p-Smad1/5/8 were found localized to primary cilia. Accompanying the resorption of primary cilia following the cells being exposed to simulated microgravity, the expression levels of these signalling proteins were reduced significantly. Furthermore, the expression of miRNA-129-3p, a microRNA previously reported to control cilium biogenesis, was found to be reduced quickly and changed in a similar tendency with the length of primary cilia. Moreover, overexpression of miRNA-129-3p in ROBs significantly attenuated microgravity-induced inhibition of BMP-2 signalling and loss of osteogenic differentiation and mineralization. These results indicated the important role of miRNA-129-3p in microgravity-induced resorption of primary cilia of osteoblasts and the potential of replenishing the miRNA-129-3p as an effective countermeasure against microgravity-induced loss of primary cilia and impairment of osteoblast function.

[The effects of different herbal compound and extracts from different extraction methods on hypoxia tolerance in mice].

Objective: To investigate the effects of different prescription compositions of traditional Chinese medicine and its different extraction methods of compound formula extracts on hypoxia tolerance in mice, in order to preferably select their prescription compositions and preparation extraction methods. Methods: Male BALB/c mice were randomly divided into 6 groups: blank control group, compound danshen group, compound Rhodiola Rosea alcohol-water extract group (Rhodiola rosea, Astragali Radix, Polygonati Rhizoma, Lycii Fructus), compound Rhodiola Rosea water extract group, compound Astragalus alcohol-water extract group (Astragali Radix, Polygonati Rhizoma, Lycii Fructus) and compound Astragalus water extract group, 30 mice in each group. Each group was administered continuously by gavage for 10 d. The blank group was gavaged with sterilized injection water. The mice in the other groups were treated with 0.15 g/kg of compound danshen, 3 g/kg of compound Rhodiola Rosea alcohol-water extract or water extract, and 1.7 g/kg of compound Astragalus alcohol-water extract or water extract, respectively. Each group was subjected to normobaric hypoxia tolerance test, sodium nitrite toxicity survival test and acute cerebral ischemia-hypoxia test 1 h after the last gavage, and the mice brain tissues were used to determine the activity of antioxidant enzymes and metabolites related to oxidative stress. Results: Compared with the blank control group, in normobaric hypoxia tolerance test, the survival time of mice in the compound danshen group and the compound Astragalus alcohol-water extract group and water extraction group was prolonged significantly (P＜0.01), and the number of open-mouth gasping after cerebral ischemia and hypoxia was increased significantly (P＜0.05). There was no statistical difference in survival time after sodium nitrite injection in each group. Compared with the blank control group, the activities of T-AOC, SOD, GSH and CAT were increased significantly (P＜0.05, P＜0.01) and the content of MDA was decreased significantly (P＜0.01) in the compound Astragalus water extract group. Compared with the compound danshen group, the activities of SOD, CAT and GSH were increased significantly (P＜0.01, P＜0.05) and the content of MDA was decreased significantly (P＜0.05). Conclusion: Compound Astragalus water extraction has the best effect of hypoxia tolerance, compound Rhodiola Rosea can eliminate Rhodiola rosea and consists of Astragali Radix, Polygonati Rhizoma, Lycii Fructus and its extraction method is water extraction.

The interdependent relationship between the nitric oxide signaling pathway and primary cilia in pulse electromagnetic field-stimulated osteoblastic differentiation.

Pulsed electromagnetic fields (PEMFs) have long been recognized being safe and effective in treating bone fracture nonunion and osteoporosis. However, the mechanism of osteogenic action of PEMFs is still unclear. While primary cilia are reported to be a sensory organelle for PEMFs, and nitric oxide (NO) plays an indispensable role in osteogenic effect of PEMFs, the relationship between NO and primary cilia is unknown. In this study, effects of treatment with 50 Hz 0.6 mT PEMFs on osteogenic differentiation and mineralization, NO secretion, and ciliary location of specific proteins were examined in rat calvarial osteoblasts (ROBs) with normal or abrogated primary cilia. It was found that PEMFs stimulated the osteogenic differentiation by activating the NOS/NO/sGC/cGMP/PKG signaling pathway, which need the existence of primary cilia. All components of the signaling pathway including iNOS, eNOS, sGC, PKG-1, and PKG-2 were localized to primary cilia, and eNOS was phosphorylated inside the primary cilia. Besides, primary cilia were elongated significantly by PEMF treatment and changed dynamically with the activation NO/cGMP pathway. When the pathway was blocked by L-NAME, PEMFs could no longer elongate the primary cilia and stimulate the osteoblastic differentiation. Thus, this study for the first time observed activation of the NO/cGMP signaling pathway in ciliary compartment of osteoblasts, and PEMFs could not stimulate the osteoblastic differentiation if the NO signaling pathway was blocked or the ciliogenesis was inhibited. Our findings indicate the interdependent relationship between NO and primary cilia in the PEMF-promoted osteogenesis.

LRP8, modulated by miR-1262, promotes tumour progression and forecasts the prognosis of patients in breast cancer.

This research was designed to detect the function of low-density lipoprotein receptor (LDLR)-related protein 8 (LRP8) in breast cancer (BC). Our results revealed that LRP8 was highly expressed in BC tissues and cell lines compared with human normal breast tissues. The poor prognosis of patients with BC was associated with the up-regulation of LRP8 while inversely connected with overexpression of miR-1262. Functionally, LRP8 depletion in BC cells impaired the proliferative, clonogenic, invasive, and migratory capabilities, which was consistent with the effects of upregulated miR-1262. Bioinformatics prediction and luciferase reporter assay confirmed that miR-1262 was an upstream factor for LRP8 and negatively regulated the expression of LRP8. Further experiments illustrated that the co-transfection of miR-1262 antamir and si-LRP8 could significantly suppress the promoting impacts caused by the transfection of miR-1262 antamir alone. These findings highlighted that LRP8 accelerated the BC development by contributing cellular aggressiveness, which was modulated by miR-1262.

miRNA-223 is an anticancer gene in human non-small cell lung cancer through the PI3K/AKT pathway by targeting EGFR.

The present study aimed to further explore the molecular mechanisms of miRNA-223 in non-small cell lung cancer (NSCLC). Data prospectively collected from NSCLC patients and volunteers from March 2016 to MayMAP2016 at Tsinghua Changgung Hospital were analyzed. Cell proliferation was measured using MTT assay, while cell apoptosis and caspase-3/9 activity were measured using flow cytometry and caspase-3/9 activity kit. Bax, EGFR, PI3K and p-Akt protein were also investigated using western blotting. The results revealed that the serum levels of miRNA-223 in NSCLC patients were downregulated. In an inMAPvitro model, over-expression of miRNA-223 induced apoptosis while reducing cell proliferation. In contrast, downregulation of the expression of miRNA-223 inhibited apoptosis whereas it increased cell proliferation. Meanwhile, overexpression of miRNA-223 suppressed the protein expression of EGFR, PI3K and p-Akt in NSCLC cells. An EGFR inhibitor promoted the anticancer effects of miRNA-223 in NSCLC cells through the EGFR/PI3K/AKT pathway. Meanwhile, a PI3K inhibitor increased the anticancer effects of miRNA-223 in NSCLC cells through the PI3K/AKT pathway. Thus, a new pathway has been identified in the present study, and application of miRNA-223 may induce the apoptosis of NSCLC through the PI3K/AKT pathway by EGFR.

Sinusoidal Electromagnetic Fields Increase Peak Bone Mass in Rats by Activating Wnt10b/ß-Catenin in Primary Cilia of Osteoblasts.

Extremely low-frequency electromagnetic fields have been considered a potential candidate for the prevention and treatment of osteoporosis; however, their action mechanism and optimal magnetic flux density (intensity) parameter are still elusive. The present study found that 50-Hz sinusoidal electromagnetic fields (SEMFs) at 1.8 mT increased the peak bone mass of young rats by increasing bone formation. Gene array expression studies with femoral bone samples showed that SEMFs increased the expression levels of collagen-1α1 and Wnt10b, a critical ligand of the osteogenic Wnt/ß-catenin pathway. Consistently, SEMFs promoted osteogenic differentiation and maturation of rat calvarial osteoblasts (ROBs) in vitro through activating the Wnt10b/ß-catenin pathway. This osteogenesis-promoting effect of SEMFs via Wnt10b/ß-catenin signaling was found to depend on the functional integrity of primary cilia in osteoblasts. When the primary cilia were abrogated by small interfering RNA (siRNA) targeting IFT88, the ability of SEMFs to promote the osteogenic differentiation of ROBs through activating Wnt10b/ß-catenin signaling was blocked. Although the knockdown of Wnt10b expression with RNA interference had no effect on primary cilia, it significantly suppressed the promoting effect of SEMFs on osteoblastic differentiation/maturation. Wnt10b was normally localized at the bases of primary cilia, but it disappeared (or was released) from the cilia upon SEMF treatment. Interestingly, primary cilia were elongated to different degrees by different intensities of 50-Hz SEMFs, with the window effect observed at 1.8 mT, and the expression level of Wnt10b increased in accord with the lengths of primary cilia. These results indicate that 50-Hz 1.8-mT SEMFs increase the peak bone mass of growing rats by promoting osteogenic differentiation/maturation of osteoblasts, which is mediated, at least in part, by Wnt10b at the primary cilia and the subsequent activation of Wnt/ß-catenin signaling. © 2019 American Society for Bone and Mineral Research.

[Effect of Compound Medicine of Tanshinone 2A and Resveratrol on Peak Bone Mass in Growing Rats].

Objective To study the effect of the compound medicine of tanshinone 2A and resveratrol on peak bone mass in growing rats and to explore its possible mechanism,so as to explore anti-osteoporosis mechanisms of new traditional Chinese medicine (TCM) drugs. Methods Totally 40 1-month-old female Wistar rats were randomly divided into tanshinone 2A group,resveratrol group,compound group (tanshinone 2A and resveratrol),and normal control group,with 10 rats in each group. Body weight was measured once every two weeks,and the whole body bone mineral density was measured with dual-energy X-ray monthly. When the whole-body bone mineral density became statistically significant between medication groups and control group,all animals were sacrificed to determine the bone mineral density of vertebrae and right femoral bone. The biomechanical properties of femur and vertebrae were measured by AGS-X series universal test,then the bone morphology was analyzed with Fuchsin picric acid staining. Finally,the levels of tartrate-resistant acid phosphatase 5b and osteocalcin were measured with enzyme-linked immunosorbent assay.Results The body weights were not statistically significant among all groups (P>0.05). The whole-body bone mineral density showed no significant difference (P>0.05) after feeding for 1 month;however,two months later,it was significantly different between medication groups and control group;in particular,the whole-body (P=0.016),femoral (P=0.001),and vertebral bone mineral density (P=0.034),bone trabecular number (P=0.024),thickness (P=0.040),and area (P=0.038) were significantly increased in the compound group,along with the significantly decreased trabecular separation degree (P=0.032). Compared with the control group,the compound group had significantly increased osteocalcin (P=0.033) and tartrate-resistant acid phosphatase 5b (P=0.028) levels in serum.Conclusion The compound of tanshinone 2 A and resveratrol can improve the bone density and bone quality in rats,and such effect is higher than either tanshinone 2 A monomer or resveratrolmonomer.

[Effect of Xianling Gubao capsule on bone metabolism in ovariectomized rats].

To investigate the effect of Xianling Gubao capsule in preventing postmenopausal osteoporosis, forty-eight female Wistar rats were randomly divided into four groups: sham group (Sham), ovariectomized group (OVX), ethinylestradiol group (EE) and Xianling Gubao capsule group (XLGB). Rats in each group received ovariectomy, except for sham group. The XLGB group received Xianling Gubao capsule at the dose of 378 mg·kg⁻¹·d⁻¹. The dosage of EE group was 200 µg·kg⁻¹·d⁻¹, and OVX and Sham groups were only fed with equal volume of distilled water. All of the rats were put to death two months later. Bone mineral density, bone biomechanics, bone histomorphometry Micro-CT scanning and organ index of vital organs were calculated and pathologically observed. There was no significant difference in the body weight of rats and organ indexes of lung, kidney, heart and spleen in the experimental groups. There was also no significant change in their pathological observation, but the uterine index of OVX group and XLGB group was significantly lower than that of Sham group. According to the results of BMD test, compared with the OVX group, femurs and vertebrae BMD of the other three groups were increased, with statistically significant differences. On the basis of the results of bone biomechanical test, compared with OVX group, the maximum load values of femur and vertebrae of the other three groups were increased, with statistically significant differences, while the change of elastic modulus was not statistically significant. According to the bone histomorphometry results of VG staining, compared with Sham group, the number of trabecular bone was significantly lower than that in OVX group. Compared with OVX group, the number of trabecular bone in EE group and XLGB group was increased, but with no significant difference between EE and XLGB groups. The results of serum biochemical indexes showed that compared with Sham group, osteocalcin (OC) decreased, while tartrate resistant acid phosphatase 5b (TRACP 5b) increased in OVX group, with statistically significant differences. Compared with OVX group, the OC content of XLGB group and EE group increased, while the content of TRACP 5b decreased, with statistically significant differences. On the basis of the results of Micro-CT scanning, the change trends of femur volume BMD, number of trabecular bone (Tb.N), trabecular bone thickness (Tb.Th), trabecular bone separation (Tb.Sp), bone volume/tissue volume (BV/TV) in the groups were consistent with those of bone histomorphometry. There was no significant change in femoral cortical bone between the two groups. Xianling Gubao capsule can prevent osteoporosis in ovariectomized rats. The possible mechanism is the dual activity of inhibiting bone resorption and improving bone formation.

[Resveratrol and puerarin on peak bone mass in young rats].

OBJECTIVE: To compare effects of resveratrol, puerarin and the compounds on peak bone mass in rats. METHODS: Forty SPF Wistar rats weighed 109.45 g to 119.44 g with an average of 115.87 g were selected. After 3 days' adaption, rats were divided into control group (the same volume of distilled water per day), puerarin group(15.4 mg/kg puerarin daily), resveratrol group (8.4 mg/kg resveratrol daily), compound drug group (daily dose of 8.4 resveratrol added 15.4 mg/kg of puerarin) and 10 in each group. The body weight of the rats was monitored at every 7 days and body bone density was measured at every month. All rats were sacrificed after 3 months. The bone mineral density of femur and vertebrae was detected by dual energy X-ray absorptiometry; bone biomechanics, VG staining was used to analyze bone histomorphometry;ELISA was used to detect serum bone metabolic index and microstructure of femur were scanned with Micro-CT scanner. RESULTS: There were no significant differences in body weight among groups during exoeriment. Bone mineral density results showed BMD of femur and vertebrae in the other three groups were significantly increased, and R+P group was significantly higher than PR group and RES group(P<0.05) by compared with CON group;three-point bending and compression test results showed compared with CON group, other three groups of femoral and vertebral maximum load values were significantly increased, and P+R group was higher than PR group and RES group, but elastic modulus was not statistically significant. Bone histomorphometry showed that number of trabecular bone in other three groups were significantly increased compared with CON group, separation of trabecular bone were significantly reduced, continuity was improved, and R+P group was significantly better than RES and PR group. The results of Micro-CT scan showed that separation of trabecular bone were significantly reduced, continuity were improved in other three groups, and R+P group was significantly better than RES and PR group. The numbers of trabecular bone (Tb.N), trabecular bone thickness (Tb.Th), volume of trabecular bone (BV/TV) in PR group, RES group and R+P group were significantly higher than CON group, but trabecular bone separation (Tb.Sp) was significantly reduced. Serum levels results showed, level of OC in the other three groups were higher than control group(P<0.05), content of TRACP 5b decreased, and level of OC in P+R group was significantly higher than PR group and RES group, content of TRACP 5b was no significant change. CONCLUSIONS: Compound of puerarin and resveratrol assigned in a 1:1 ratio could improve bone mineral density and bone mass in young rats, enhance biomechanical properties of bone, promote mineralization and maturation of osteoblasts, inhibit osteoblastic bone resorption, and is better than the role of their respective monomers. The paper showed that traditional Chinese medicine compound medicine will be used as a new way to prevent and treat osteoporosis.

Total flavonoid extract of Epimedium herb increases the peak bone mass of young rats involving enhanced activation of the AC10/cAMP/PKA/CREB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Epimedium sagittatum brevicornum Maxim. is an important traditional Chinese herb that has long been used to promote bone fracture healing and treat osteoporosis. AIM OF THE STUDY: Achieving peak bone mass by adolescence has now been accepted to be fundamental for preventing osteoporosis in adulthood life. This study investigated the possibility of increasing peak bone mass in young rats using the total flavonoid extract of Epimedium herb (TFE). MATERIALS AND METHODS: TFE was intragastrically administered to one-month-old Wistar rats at a low (100 mg/kg), middle (200 mg/kg) or high dose (400 mg/kg). Whole body bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry every two weeks. When BMD of any one of TFE groups was found to be significantly higher than that of the control, all rats were sacrificed, serum samples were collected for bone turnover biochemical assays, and femurs, tibiae and vertebrae were isolated and used in BMD, mechanical, micro-structural, histomorphometric and mechanistic studies. RESULTS: Administration of TFE at middle and high doses for two months significantly increased the whole body, femoral and vertebral BMDs, and improved the bone mechanical and micro-architectural properties. The serum turnover biochemical results and the enhanced expression levels of bone-formation regulatory genes (Runx-2, OSX, and BMP-2) demonstrated that TFE administration increased bone formation but had no effect on bone resorption. The increased phosphorylation levels in femurs of PKA and CREB and expression of AC10 (the only soluble form of adenylyl cyclase) and the increased serum cAMP level after 4 h of TFE administration indicated that TFE promoted bone formation by activating the AC10/cAMP/PKA/CREB pathway in vivo. CONCLUSIONS: Oral administration of TFE at 200 mg/kg for two months can increase the peak bone mass of growing rats, suggesting the possibility of using total flavonoid extract of Epimedium herb to increase the peak bone mass in adolescence which is important for preventing osteoporosis in adult life.

Pulsed electromagnetic fields stimulate osteogenic differentiation and maturation of osteoblasts by upregulating the expression of BMPRII localized at the base of primary cilium.

Pulsed electromagnetic fields (PEMFs) have been considered as a potential candidate for the prevention and treatment of osteoporosis, however, the mechanism of its action is still elusive. We have previously reported that 50Hz 0.6mT PEMFs stimulate osteoblastic differentiation and mineralization in a primary cilium- dependent manner, but did not know the reason. In the current study, we found that the PEMFs promoted osteogenic differentiation and maturation of rat calvarial osteoblasts (ROBs) by activating bone morphogenetic protein BMP-Smad1/5/8 signaling on the condition that primary cilia were normal. Further studies revealed that BMPRII, the primary binding receptor of BMP ligand, was readily and strongly upregulated by PEMF treatment and localized at the bases of primary cilia. Abrogation of primary cilia with small interfering RNA sequence targeting IFT88 abolished the PEMF-induced upregulation of BMPRII and its ciliary localization. Knockdown of BMPRII expression level with RNA interference had no effects on primary cilia but significantly decreased the promoting effect of PEMFs on osteoblastic differentiation and maturation. These results indicated that PEMFs stimulate osteogenic differentiation and maturation of osteoblast by primary cilium-mediated upregulation of BMPRII expression and subsequently activation of BMP-Smad1/5/8 signaling, and that BMPRII is the key component linking primary cilium and BMP-Smad1/5/8 pathway. This study has thus revealed the molecular mechanism for the osteogenic effect of PEMFs.

A new anti-fibrinolytic hemostatic compound 8-O-acetyl shanzhiside methylester extracted from Lamiophlomis rotata.

BACKGROUND: Fibrinolysis prevents blood clots from growing and becoming problematic. Antifibrinolytics are used as inhibitors of fibrinolysis. Aprotinin was doubted after identification of major side effects, especially on kidney. Lysine analogues has their own defects and whether they are adequate substitutes for aprotinin is still under doubt. Lamiophlomis rotata (Benth.) Kudo. was previous found to have hemostatic activity. But the active compound in L. rotata and its hemostatic mechanism were unknown. OBJECTIVES: To find the major hemostatic compound in L. rotata and identify its haemostasis mechanism. METHODS: Traumatic hemorrhage model and coagulant activity assays were monitored in mice and platelets in drug treatment group and control group. Hyperfibrinolysis model was established by intravenous administration of urokinase in mice. Capillary blood clotting time (CBCT), activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen and euglobulin clot lysis time (ECLT) were measured. RESULTS: The anti-fibrinolytic activity come from 8-O-Acetyl shanzhiside methylester (ASM) one of the highest iridoid glycosides contents in TIG extracted from L. rotata. ASM significantly (P<0.05) shorten CBCT and reduced blood loss volume in vivo, but did not influence mice APTT, PT or TT. In particular, it significantly prolonged ECLT in hyperfibrinolysis mice. It indicated that ASM could inhibit fibrinolysis. ASM was also effective in CBCT, traumatic bleeding volume and ECLT in hyperfibrinolysis mice model. CONCLUSIONS: ASM was the major hemostatic compound in L. rotata. The haemostasis mechanism of ASM was achieved by anti-fibrinolytic activity. ASM was a new fibrinolysis inhibitor as iridoid glycoside compound.

[Protective Effect of Saussurea involucrata Alcohol Extract on Liver Mitochondria in Mice Under Hypoxia Condition].

OBJECTIVE: To study the protective effect of Saussurea involucrata alcohol extract on liver mitochondria in mice under hypoxia condition. METHODS: The hypoxia mice model was established, the BALB/c mice were randomly divided into four groups:normal group ,hypoxia model group, positive control group and Saussurea involucrata alcohol extract group. Mice were put into low pressure oxygen chamber and decompressed, adapted to hypobaric hypoxia environment of simulated altitude of 8,000 m for 12 h, and then recovered to normal altitude. The mice were sacrificed and the liver mitochondria was isolated, the mitochondrial membrane potential and the activity of malate dehydrogenase, aconitase, α-ketoglutarate dehydrogenase, pyruvate dehydrogenase and mitochondrial complex I, II and V were measured. RESULTS: Compared with hypoxia model group, Saussurea involucrata alcohol extract protected mitochondrial membrane potential, sustained the activities of aconitase, α-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and mitochondrial complex I, II and V under hypoxia condition. CONCLUSION: Saussurea involucrata alcohol extract can protect the liver mitochondrial function in mice under hypoxia condtion.

Anti-hypoxia Activity of the Novel NO Donor Acetyl Ferulic Isosorbide Mononitrate in Acute High-Altitude Hypoxia Mice.

Nitric oxide (NO) may act as either a pro-oxidant or an antioxidant in biological systems. Previous work has found inhalation of NO improved survival in a high altitude rat model. NO donor isosorbide mononitrate derivants might have a protective effect against hypoxia. We synthesized a series of isosorbide mononitrate derivant compounds to test their anti-hypoxia activities. Normobaric hypoxia and hypobaric hypoxia models were used to study the protective role of NO donor in mice. The results showed isosorbide mononitrate derivants had protective effects in hypoxia mice. Among those compounds, acetyl ferulic isosorbide mononitrate (AFIM) was the most effective. It prolonged the survival time during the normobaric hypoxia test. It decreased malondialdehyde (MDA) and H2O2 in hypobaric hypoxia mice. The antioxidase activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) remained in normal ranges in the AFIM group. As a sign of mitochondrial dysfunction, the activities of ATPase were down regulated in mice under hypobaric hypoxia conditions. AFIM also protected ATPase activities. The protective effects of AFIM might come from a sustained NO supply and the release of acetyl ferulic acid with anti-oxidant activity.

[Establishment of osteoblast primary cilia model removed by chloral hyrate].

OBJECTIVE: To establish osteoblast model, primary cilla model was removed by chloral hyrate, observe effects of osteoblast primary cilla moved on enhancing ALP staining and calcified nodules staining in electromagnetic field. METHODS: Three 3-day-old male SD rats weighed between 6 and 9 g were killed, cranial osteoblast was drawed and adherencing cultured respectively. Cells were subcultured and randomly divided into 4 groups until reach to fusion states. The four groups included chloral hydrate non-involved group (control group), 2 mM, 4 mM and 8 mM chloral hydrate group, and cultured in 37 °C, 5% CO2 incubator for 72 h. Morphology of primary cilla was observed by laser confocal scanning microscope, and incidence of osteoblast primary cilia was analyzed by Image-Pro Plus 6.0 software. Cells in the correct concentration group which can removed cillia most effectively were selected and divided into 3 groups, including control group (C), Electromagnetic fields group (EMFs), and EMFs with 4 mM chloral hydrate group. DMEM nutrient solution contained 10%FBS were added into three groups and cultured for 9 days and formation of ALP were observed by histochemical staining of alkaline phosphatase. After 12 days' cultivation, formation of mineralization nodes was observed by alizarin red staining. RESULTS: Compared with control group and 2mM chloral hydrate group,4 mM chloral hydrate group could effectively remove osteoblast primary cilla (P<0.01). Removal of osteoblast primary cilla could weaken the formation of ALP and mineralization nodes in osteoblast in EMFS. Compared with EMFs group, the area of ALP and mineralization nodes in EMFs with 4 mM chloral hydrate group were decreased obviously (P<0.01). CONCLUSION: 4mM chloral hydrate could effectively remove osteoblast primary cilia. Primary cilla participate in EMFs promoting formation of ALP and mineralization nodes in osteoblast and provide new ideas for exploring mechanism of EMFs promoting osteoblast maturation and mineralization.

[Chemical Constituents with Anti-hypoxia Activity from Saussurea involucrata].

OBJECTIVE: To investigate the chemical constituents with anti-hypoxia activity from Saussurea involucrata. METHODS: The chemical constituents, isolated and purified by column chromatography from Saussurea involucrata, were identified by several spectroscopic methods. The anti-hypoxic activities of these compounds were examined using the normobaric hypoxic model of mice. RESULTS: Twelve compounds were isolated from petroleum ether extract of Saussurea involucrata and identified as n-octacosane (1), 1-undecanol (2), heptadecan-l-ol(3), heptacosan-1-ol(4), myristicin (5), apiol(6), ß-sitosterol(7), lupeol(8), moslosooflavone (9), mosloflavone (10), negletein(11), and 5, 6-dihydroxy-7, 8-dimethoxyflavone(12). CONCLUSION: All compounds except 7 and 8 are isolated from this plant for the first time. Compound 1, 5 and 8 - 12 can significantly prolong the survival time of hypoxic mice.

Convergent synthesis of moslosooflavone, isowogonin and norwogonin from chrysin.

A convergent synthesis route of moslooflavone, isowogonin and norwogoninis reported,starting from chrysin, an easily available flavone, by methylation, bromination, methoxylation and demethylation procedures. This synthetic route is convenient and can give the three rare flavones in good yield.

Pulsed electromagnetic fields promote osteoblast mineralization and maturation needing the existence of primary cilia.

Although pulsed electromagnetic fields (PEMFs) have been approved as a therapy for osteoporosis, action mechanisms and optimal parameters are elusive. To determine the optimal intensity, exposure effects of 50 Hz PEMFs of 0.6-3.6 mT (0.6 interval at 90 min/day) were investigated on proliferation and osteogenic differentiation of cultured calvarial osteoblasts. All intensity groups stimulated proliferation significantly with the highest effect at 0.6 mT. The 0.6 mT group also obtained the optimal osteogenic effect as demonstrated by the highest ALP activity, ALP(+) CFU-f colony formation, nodule mineralization, and expression of COL-1 and BMP-2. To verify our hypothesis that the primary cilia are the cellular sensors for PEMFs, osteoblasts were also transfected with IFT88 siRNA or scrambled control, and osteogenesis-promoting effects of 0.6 mT PEMFs were found abrogated when primary cilia were inhibited by IFT88 siRNA. Thus primary cilia of osteoblasts play an indispensable role in mediating PEMF osteogenic effect in vitro.

[Comparative study on effect of osthole and genistein on peak bone mass in rats].

OBJECTIVE: To compare the ability of osthole (OST) and genistein (GEN) in enhancing bone peak bone mass of rats to prevent osteoporosis. METHODS: Thirty-six female one-month-old SD rats of (125 +/- 3) g body weight were randomly divided into three groups, 12 rats in each group, one group was orally administered osthole at 9 mg x kg(-1) d(-1), one group was given genistein at 10 mg x kg(-1) d(-1) and another was given equal quantity of distilled water as the control. The body weight was monitored weekly and the bone mineral density (BMD) of total body was measured every month. All rats were sacrificed after three months, the femoral bone mineral density, the serum levels of osteocalcin (OC) and anti-tartaric acid phosphatase 5b (TRACP 5b) were measured by Elisa. The bone microarchitectures were analyzed with micro-CT and the bone biomechanics properties were tested with universal material machine. RESULTS: No significant differences were observed between O-treated or GEN group and the control for the food-intake and body weight during three months. However, the rats treated with OST had significant higher BMD for both total body and femur than the control and GEN group. The O-treated rats also had higher level of serum OC and lower level of TRACP 5b. Besides, they owned bigger bone volume/tissue volume, trabecular thickness, trabecular number but smaller trabecular spacing. In the three point bending tests of femurs,they were found to have larger maximum load, the young's modulus and structural model index (SMI). CONCLUSION: Orally administered osthole could efficiently increase the peak bone mass of rats,which provide new ideas for preventing osteoporosis.