Comparison of the Efficacy of Indwelling Gastric Tubes in Preoperative and Postoperative Patients With Oral and Maxillofacial Malignancies.

PURPOSE: To explore the optimal plan for the timing of indwelling gastric tube placement in oral and maxillofacial malignant tumor patients. DESIGN: A prospective randomized controlled trial. METHODS: 80 patients with oral and maxillofacial tumor were selected, and 40 patients were Pre-operative group. The remaining 40 patients were the control group, called Postoperative group. The body weight and hospital stay of the two groups were observed before and after surgery. Blood samples were taken before surgery and 1, 3 and 7 days after surgery to detect hemoglobin and plasma albumin. FINDINGS: The number of postoperative hospitalization days in the pre-operative group was significantly lower than that in the post-operative group; postoperative hemoglobin and plasma albumins were lower in both groups compared with the preoperative level. CONCLUSIONS: Preoperative nasogastric tube ensured early postoperative administration of gastrointestinal nutrition, promoted postoperative plasma albumin recovery, and shortened the days of hospitalization.

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.

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.

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.

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.

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.

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.

[Comparison research with icariin and genistein by anti-inflammatory reaction and angiogenesis pathway to inhibit bone loss on ovariectomized rats].

OBJECTIVE: Through researching the relationship among osteoporosis and inflammatory reaction besides angiogenesis, to compare pharmacological differences between icariin and genistein to inhibit bone loss. METHODS: 6 months old female SD rats were randomly divided into SHAM group, model group, ICA group, GEN group and E group. The bone mineral density of total, femur and lumbar, serum OC, TRACP 5b, IL-6 and VEGF, biomechanics of femur and tibia microarchitecture were analyzed. RESULTS: Compared with SHAM group, model group of body weight, uterine weight, bone mineral density of total, femur and lumbar, serum OC, TRACP 5b, IL-6 and VEGF, biomechanics of femur and lumbar and tibia microarchitecture were significantly changed (P < 0.05). Compared with model group, ICA group of body weight, bone mineral density of total and femur, serum TRACP 5b and femural biomechanics were significantly changed (P < 0.05). GEN group of bone mineral density of total, femur and lumbar, serum OC, TRACP 5b, IL-6 and VEGF, biomechanics of femur and lumbar and tibia microarchitecture were significantly changed (P < 0.05). CONCLUSION: Icariin inhibits bone loss on model rat through suppressing bone resorption. Genistein prevents bone loss on model rat by the pathway of inhibiting inflammatory reaction, activating angiogenesis, enhancing bone formation and inhibiting bone resorption. Moreover, pharmacological activity of genistein is more potential than icariin.

[Protective effect and action mechanism of petroleum ether extracts from Saussurea involucrate on brain tissues of hypoxia rats].

OBJECTIVE: To investigate the protective effect and action mechanism of petroleum ether extracts from Saussurea involucrate on brain tissues of hypoxia rats under constant pressure and closed conditions. METHOD: The PESI dosage-dependent experiment for hypoxia rats was conducted under constant pressure and closed conditions by intraperitoneally injecting 125, 250, 500 mg x kg(-1) to finalize that the optimum dosage is the high dose of PESI. Afterwards, 90 Wistar rats were randomly divided into the hypoxic model group, the acetazolamide 250 mg x kg(-1) group and the PESI high dose group. Each group was further divided into three subgroups according to different hypoxia times, with 10 rats in each subgroup. Under the same hypoxia and administration conditions, the rats were sacrificed after 0, 3, 6 h respectively. Their brain samples were collected for common pathological observation and immunohistochemical staining of HIF-1alpha. Real-time RT-PCR was used to detect HIF-1alpha, EPO, HO-1 and Caspase-3 gene expressions. And the Western blot assay was adopted to detect HIF-1alpha protein expression. RESULT: The brain tissues of the hypoxia model group were severely damaged with the increase in the hypoxia time. The acetazolamide group and the PESI high does group were damaged in a much lower degree. According to the gene expression and the Western blot assay, high dose of PESI could inhibit HIF-1alpha expression. According to the pure gene expression test, high dose of PESI could increase EPO and HO-1 mRNA expressions, but inhibit Caspase-3 mRNA expression. CONCLUSION: PESI's protective mechanism for brain tissues of hypoxia rats under constant pressure and closed conditions may be related to its effects in inhibiting HIF-1alpha expression, increasing EPO expression and resisting cell apoptosis.

Icariin stimulates the osteogenic differentiation of rat bone marrow stromal cells via activating the PI3K-AKT-eNOS-NO-cGMP-PKG.

Icariin, a prenylated flavonol glycoside isolated from Epimedii herba, has been found to be a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of icariin is related to the nitric oxide (NO) signal pathway and PI3K/AKT pathway in its upstream. Rat bone marrow stromal cells (rBMSCs) were cultured in osteogenic medium and treated with icariin or together with L-NAME, ODQ, PDE5, and/or LY294002 (the inhibitor of NOS, sGC, cGMP, and PI3K respectively), and effects were examined on the expression of signal messengers (NOS, NO, sGC, cGMP, PKG and PI3K) and the levels of osteogenic markers (alkaline phosphatase or ALP, osteocalcin and calcified nodules). It was found that icariin dose-dependently increased ALP activity, and treatment at the optimal concentration (10(-5)M) increased NOS activity, iNOS and eNOS expression, NO production, sGC and cGMP contents and PKG expression besides the phosphorylation of AKT. The addition of L-NAME, ODQ and PDE5 significantly inhibited the icariin effects on above markers respectively. The addition of LY294002 decreased the p-AKT level, NOS activity, eNOS expression and NO production significantly, but had no significant effect on iNOS expression. The addition of any of the four inhibitors also abolished the osteogenic effect of icariin on rBMSCs as indicated by ALP activity, osteocalcin synthesis, calcium deposition and the number and areas of calcified nodules. These results suggest that the osteogenic effect of icariin involves the PI3K-AKT-eNOS-NO-cGMP-PKG signal pathway. Furthermore, dosage response studies showed that icariin at 10(-6)M (a physiologically achievable concentration in vivo) also activated this signal pathway.

Different electromagnetic field waveforms have different effects on proliferation, differentiation and mineralization of osteoblasts in vitro.

Noninvasive electromagnetic fields (EMFs) have been known to be able to improve bone health; however, their optimal application parameters and action mechanisms remain unclear. This study compared the effects of different forms of EMFs (sinusoidal, triangular, square, and serrated, all set at 50 Hz frequency and 1.8 mT intensity) on proliferation, differentiation and mineralization of rat calvarial osteoblasts. Square EMFs stimulated osteoblast proliferation but sinusoidal EMFs inhibited it. Sinusoidal and triangular EMFs produced significantly greater alkaline phosphatase (ALP) activity, ALP staining areas, calcium deposition, mineralized nodule areas, and mRNA expression of Runx-2, osteoprotegerin and insulin-like growth factor-I than square and serrated EMFs (P < 0.01). Triangular EMFs had a greater effect than sinusoidal EMFs on every indices except for Runx-2 mRNA expression (P < 0.05). These results indicated that while square EMFs promoted proliferation and had no effect on the differentiation of osteoblasts, sinusoidal EMFs inhibited proliferation but enhanced osteogenic differentiation. Triangular EMFs did not affect cell proliferation but induced the strongest osteogenic activity among the four waveforms of EMFs. Thus, the effects of EMFs on proliferation and differentiation of osteoblasts in vitro were dependent on their waveforms.

The prenyl group contributes to activities of phytoestrogen 8-prenynaringenin in enhancing bone formation and inhibiting bone resorption in vitro.

Previous studies have found that 8-prenylflavonoids have a higher osteogenic activity than do flavonoids, which suggested that the 8-prenyl group may play an active role in bone-protective properties. To address this hypothesis, activities of 8-prenylnaringenin (PNG) and naringenin (NG) in osteoblast and osteoclast differentiation and function were compared in vitro. PNG was found to have a stronger ability than NG to improve osteoblast differentiation and osteogenic function in cultured rat calvarial osteoblasts, as demonstrated by levels of alkaline phosphatase activity, osteocalcin, calcium deposition, and the number and area of mineralized bone nodules, as well as mRNA expression of osteogenesis-related genes Bmp-2, OSX, and Runx-2. In addition, although expression of osteoclastogenic inducer receptor activator of nuclear factor kappa-B ligand (RANKL) was not affected, that of osteoclastogenesis inhibitor osteoprotegerin (OPG) and consequently the OPG/RANKL ratio were increased, more potently by PNG than NG. PNG was also found to have a higher potency than NG in inhibiting the osteoclast formation in rabbit bone marrow cells and their resorptive activity, as revealed by lower numbers of osteoclasts formed, lower numbers and areas of bone resorption pits, and lower mRNA expression levels of tartrate-resistant acid phosphatase and cathepsin K. Furthermore, PNG induced apoptosis of mature osteoclasts at a higher degree and at an earlier time than did NG. These results indicate that the 8-prenyl group plays an important role and contributes to the higher bone-protective activity of PNG in comparison with NG.

[Effects of chlorogenic acid on the viability and HIF-1alpha mRNA expression of PC12 cells exposed to hypoxia].

OBJECTIVE: To investigate the effects of chlorogenic acid on the viability and HIF-1alpha mRNA expression of PC12 cells exposed to hypoxia. METHODS: PC12 cells were cultured in trigas incubator in order to establish the hypoxic condition. The effects of chlorogenic acid on the cells were evaluated by morphological observation, cell viability and LDH release assays as well as the examination of mRNA expression level of HIF-1alpha. RESULTS: Chlorogenic acid significantly improved the viability of cells exposed to hypoxia, decreased LDH release, arrested the cell cycle on G1 phase, and increased the gene expression level of HIF-1alpha. CONCLUSION: Chlorogenic acid protects PC12 cells from hypoxic damage by improving the expression of HIF-1alpha.

[Comparison of effects of kaempferide and anhydroicaritin on biomineralization of cultured osteoblasts].

This study is to compare the effects of kaempferide and anhydroicaritin on biomineralization of rat osteoblasts (ROB) in vitro. Calvarias were dissected aseptically from newborn SD rats, the osteoblasts were obtained by enzyme digestion and were cultured in MEM containing 10% FBS. The medium was changed every three days, and serial subculture was performed when cells covered with 90% of the dish. Kaempferide and anhydroicaritin were separately added with final concentrations of 1 x 10(-4), 1 x 10(-5), 1 x 10(-6) and 1 x 10(-7) mol x L(-1) under the conditions of osteogenic differentiation. The proliferation was measured by MTT, and the optimal concentration was detected by the ALP activity at the 9th day after osteogenic induction culture. The osteogenic indexes of kaempferide, anhydroicaritin and control group with the optimal concentration were compared. The result showed that the anhydroicaritin at concentration of 1 x 10(-5) mol x L(-1) had significantly promoted the activity of ALP, calcium content and osteocalcin content, increased the number of CFU-F(ALP) and mineralized nodules, enhanced the mRNA level of BMP-2, OSX and Runx-2, which are key genes of osteogenic differentiation, and raised the protein content of collagen-I. However, the kaempferide group had not significantly represented the ability that promoted osteogenic differentiation of ROB. The difference of osteogenic differentiation on ROB between kaempferide and anhydroicaritin was caused by the prenyl group on C-8 of icariin.

Anti-hypoxic activity at simulated high altitude was isolated in petroleum ether extract of Saussurea involucrata.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhodiola algida, Saussurea involucrata, and other herbs grown in Qinghai-Tibetan plateau have long been used to prevent and treat acute mountain sickness. AIM OF THE STUDY: To screen and identify the anti-hypoxic constituents in the herbs grown in Qinghai-Tibetan plateau of Northwestern China. MATERIALS AND METHODS: The anti-hypoxic activities of 20 selected plateau herbs were examined against two positive controls, Rhodiola algida and acetazolamide, using the normobaric hypoxia model of mice. The herb with the highest activity was successively extracted with 70% ethanol, petroleum ether, chloroform, ethyl acetate and n-butanol. The extract with the highest activity was identified by comparing the survival time of mice under normobaric hypoxia condition after being subjected to different extracts. The identified extract was further tested by simulating high altitudes through an acute decompression model and a chronic decompression model for mice. RESULTS: The herb found to have the highest anti-hypoxic activity was Saussurea involucrate (Kar. et Kir.) Sch.-Bip, and the most effective fraction was in the petroleum ether extract. Administration of petroleum ether extract of Saussurea involucrata (PESI) to mice at 50mg/kg significantly decreased the mortality of animals under acute decompression conditions. Changes in biochemical indicators for glycometabolism and energy metabolism, including adenosine triphosphate (ATP) content and adenosine triphosphatase (ATPase) activity in brain and cardiac muscle, lactic acid (LAC) and lactate dehydrogenase (LDH) in blood and cardiac muscles, blood sugar, and glycogen content in liver and skeletal muscle were reversed under chronic decompression conditions. CONCLUSIONS: Saussurea involucrata (Kar. et Kir.) Sch.-Bip exhibits high anti-hypoxic activity that may be effective in preventing acute mountain sickness, and the active constituents are mainly in the petroleum ether extract.

NF-κB induces abnormal centrosome amplification by upregulation of CDK2 in laryngeal squamous cell cancer.

Centrosome amplification can drive chromosomal instability (CIN) which is a major source of tumor initiation. The present study aimed to investigate the impact of nuclear factor kappa B (NF-κB) on centrosome amplification of Hep-2 cells. Immunofluorescence was performed to display centrosomes. BAY11-7082 was used as an inhibitor of NF-κB to assess the inhibition of centrosome amplification, and cyclin-dependent kinase 2 (CDK2), ensuring cell cycle cycle coordination with centrosome cycle was detected by Western blotting. Furthermore, a 1556-bp fragment of the CDK2 promoter was analyzed using the TRANSFAC-TESS software. Luciferase assay, including a series of truncated CDK2 promoters and site mutations, was carried out to determine NF-κB binding sites in the CDK2 promoter. Electrophoresis mobility shift and chromatin immunoprecipitation assays were applied to confirm whether NF-κB indeed binds to the 5'-promoter region of the CDK2 gene. To reveal the clinical significance of CDK2 expression in laryngeal squamous cell cancer, mRNA and protein levels were assessed by RT-PCR and Western blotting, respectively. We found that the transcription factor NF-κB plays a role in centrosome amplification in Hep-2 cells. Centrosome amplification is reduced by inhibition of the NF-κB pathway. Moreover, expression of the p65 subunit of NF-κB is sufficient to promote centrosome amplification and increase in CDK2 protein levels. We further identified a functional NF-κB binding site located in the CDK2 promoter. Single mutation of the NF-κB site III (construct mutIII) however resulted in 76±5% (p<0.01) luciferase activity reduction. Electromobility shift assays and chromatin immunoprecipitaton results suggest that NF-κB indeed binds to this responsive element associating with CDK2 expression and centrosome amplification. RT-PCR and Western blotting results revealed that both mRNA and protein levels of CDK2 were significantly higher in tumor tissues than those in paired adjacent normal laryngeal tissues.

Effects of 50 Hz sinusoidal electromagnetic fields of different intensities on proliferation, differentiation and mineralization potentials of rat osteoblasts.

Electromagnetic fields (EMFs) have been used clinically to slow down osteoporosis and promote fracture healing for many years. However, the underlying action mechanisms and optimal parameters of the EMF applications are unclear. In this study, we investigated the effects of treatment for different durations with 50 Hz sinusoidal electromagnetic fields (SEMFs) at different intensities on proliferation, differentiation and mineralization potentials of rat osteoblasts. Osteoblasts isolated from neonatal rats were treated with SEMFs (50 Hz at 0.9 mT-4.8 mT, 0.3 mT interval, 30 min/day up to 15 days). Compared to untreated control, SEMFs inhibited osteoblast proliferation (after 3 days' treatment) but increased alkaline phosphatase (ALP) activity (after treatment for 9 days) from 0.9 mT to 1.8 mT, declined from 1.8 mT until 3.0 mT, and then increased again from 3.0 mT to 3.6 mT and decreased once again from 3.6 mT to 4.8 mT. Numbers of colonies stained positive for ALP after 8 days and mineralized nodules stained by Alizarin red after 10 days showed the same bimodal tendency as with the ALP activity, with two peaks at 1.8 mT and 3.6 mT. SEMFs also bimodally increased Runx-2, Col1α2 and Bmp-2 mRNA expression levels in osteoblasts at 12, 24 and 96 h after exposure. The results indicated that while exposure to 50 Hz SEMFs inhibits the osteoblast proliferation, it significantly promotes differentiation and mineralization potentials of osteoblasts in an intensity-dependent manner with peak activity at 1.8 mT and 3.6 mT.

[The changes of iNOS and NO in the osteogenic differentiation process of rat bone marrow stromal cells promoted by icariside II].

This study is to investigate the effects on the expression of iNOS and production of NO in the osteogenic differentiation process of rat bone marrow stromal cells (rBMSCs) by icariside II. rBMSCs were cultured by adherence screening method. When the culture dishes were covered with 80% cells, the osteogenic induced cultures were adopted. Icariside II was supplemented into the culture at 1 x 10(-5) mol x L(-1). The activity of iNOS, content of NO and osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-FALP and mineralized bone nodules were compared among the icariside II-supplemented group, L-NMAE group, icariside II + L-NAME group and the control. Total RNA was isolated and the gene expression of iNOS, Osterix and Runx-2 was investigated by real-time PCR. Total protein was also isolated and the secretion of iNOS and collagen I was examined by Western blotting. Icariside II can significantly improved ALP activity, CFU-FALP amount and mineralized nodules. Besides, the mRNA level of factors related to the osteogenic differentiation includes Osterix and Runx-2 also enhanced. The secretion of collagen I also promoted significantly. But all of these effects can be inhibited by L-NAME which can specifically inhibit the activity of iNOS. Icariside II enhances the osteogenic differentiation of rBMSCs significantly, but if the activity of iNOS was blocked by L-NAME, the osteogenic differentiation markers decrease accompanied with iNOS and NO decrease, suggesting that icariside II stimulates the osteogenic differentiation via enhancing the activity of iNOS and promoting the generation of NO.

Icariin is more potent than genistein in promoting osteoblast differentiation and mineralization in vitro.

There has been a strong interest in searching for natural therapies for osteoporosis. Genistein, an isoflavone abundant in soy, and icariin, a prenylated flavonol glycoside isolated from Epimedium Herb, have both been identified to exert beneficial effects in preventing postmenopausal bone loss. However, the relative potency in osteogenesis between the individual phytoestrogen flavonoids remains unknown. The present study compared ability of genistein and icariin in enhancing differentiation and mineralization of cultured rat calvarial osteoblasts in vitro. Dose-dependent studies in osteoblast differentiation measuring alkaline phosphatase (ALP) activity revealed optimal concentrations of genistein and icarrin for stimulating osteogenesis to be both at 10(-5) M. Time course studies comparing the two compounds both at 10(-5) M demonstrated that icariin treatment always produced higher ALP activity, more and larger areas of CFU-F(ALP) colonies and mineralized nodules, more osteocalcin secretion, and calcium deposition, and a higher level of mRNA expression of osteogenesis-related genes COL1α2, BMP-2, OSX, and RUNX-2. However, they inhibited the proliferation of osteoblasts to a similar degree. In conclusion, although future in vivo studies are required to investigate whether icariin is more efficient in improving bone mass and/or preventing bone loss, our in vitro studies have demonstrated that icariin has a stronger osteogenic activity than genistein. In addition, while the prenyl group on C-8 of icariin could be the active group that takes part in osteoblastic differentiation and explains its greater potency in osteogenesis, mechanisms of action, and reasons for the relative potency of icariin versus genistein need to be further studied.