Enzyme immobilized in BioMOFs: Facile synthesis and improved catalytic performance.

Biological metal-organic frameworks (BioMOFs), an emerging sub-class of MOFs, are prepared from metals and biological ligands (bioligands). Benefit from the low toxicity and good biocompatibility of bioligands, BioMOFs can be used in biomedicine and biocatalysis. In this work, a novel approach was developed for fabricating BioMOFs materials (Co-Cys BioMOFs) from cobalt salt and cystine, meanwhile nitrile hydratase (NHase) was in-situ encapsulated during the synthesis process. The obtained NHase-BioMOFs biocomposits named NHase@Co-Cys was characterized by SEM, TEM, XPS, etc. The preparation parameters and stabilities of NHase@Co-Cys were investigated. The maximum encapsulation yield and specific activity of NHase@Co-Cys were 92.71% and 139.04 U/gimmobilized NHase, respectively. The thermal stability of NHase@Co-Cys was improved by approximately 5-fold at 55 °C. The activity of NHase after immobilization was retained nearly 60% after incubating at pH 4.0 and 10.0 for 7 h. The NHase@Co-Cys showed similar catalytic capacity compared with free NHase in producing nicotinamide. After 7 h of reaction catalyzed by free NHase (14.51 U) and NHase@Co-Cys (12.76 U), the yield of nicotinamide was 90.94% and 86.36%, respectively. The activity of NHase@Co-Cys remained 83.85% of the original activity after recycling for 10 times. These results suggested that the NHase@Co-Cys is an effective approach to enhance the enzymatic properties and demonstrated a broad application prospect in industrial production.

Synaptic Homeostasis and Allostasis in the Dentate Gyrus Caused by Inflammatory and Neuropathic Pain Conditions.

It has been generally accepted that pain can cause imbalance between excitation and inhibition (homeostasis) at the synaptic level. However, it remains poorly understood how this imbalance (allostasis) develops in the CNS under different pain conditions. Here, we analyzed the changes in both excitatory and inhibitory synaptic transmission and modulation of the dentate gyrus (DG) under two pain conditions with different etiology and duration. First, it was revealed that the functions of the input-output (I/O) curves for evoked excitatory postsynaptic currents (eEPSCs) following the perforant path (PP) stimulation were gained under both acute inflammatory and chronic neuropathic pain conditions relative to the controls. However, the functions of I/O curves for the PP-evoked inhibitory postsynaptic currents (eIPSCs) differed between the two conditions, namely it was greatly gained under inflammatory condition, but was reduced under neuropathic condition in reverse. Second, both the frequency and amplitude of miniature IPSCs (mIPSCs) were increased under inflammatory condition, however a decrease in frequency of mIPSCs was observed under neuropathic condition. Finally, the spike discharge of the DG granule cells in response to current injection was significantly increased by neuropathic pain condition, however, no different change was found between inflammatory pain condition and the control. These results provide another line of evidence showing homeostatic and allostatic modulation of excitatory synaptic transmission by inhibitory controls under different pathological pain conditions, hence implicating use of different therapeutic approaches to maintain the homeostasis between excitation and inhibition while treating different conditions of pathological pain.

SDF1-CXCR4 Signaling Maintains Central Post-Stroke Pain through Mediation of Glial-Neuronal Interactions.

Central post-stroke pain (CPSP) is an intractable central neuropathic pain that has been poorly studied mechanistically. Here we showed that stromal cell-derived factor 1 (SDF1 or CXCL12), a member of the CXC chemokine family, and its receptor CXCR4 played a key role in the development and maintenance of thalamic hemorrhagic CPSP through hypoxia inducible factor 1α (HIF-1α) mediated microglial-astrocytic-neuronal interactions. First, both intra-thalamic collagenase (ITC) and SDF1 injections could induce CPSP that was blockable and reversible by intra-thalamic administration of both AMD3100 (a selective CXCR4 antagonist) and inhibitors of microglial or astrocytic activation. Second, long-term increased-expression of SDF1 and CXCR4 that was accompanied by activations of both microglia and astrocytes following ITC could be blocked by both AMD-3100 and YC-1, a selective inhibitor of HIF-1α. AMD-3100 could also inhibit release of proinflammatory mediators (TNFα, IL1ß and IL-6). Increased-expression of HIF-1α, SDF1, CXCR4, Iba1 and GFAP proteins could be induced by both ITC and intra-thalamic CoCl2, an inducer of HIF-1α that was blockable by both HIF-1α inhibition and CXCR4 antagonism. Finally, inhibition of HIF-1α was only effective in prevention, but not in treatment of ITC-induced CPSP. Taken together, the present study demonstrated that in the initial process of thalamic hemorrhagic state HIF-1α up-regulated SDF1-CXCR4 signaling, while in the late process SDF1-CXCR4 signaling-mediated positive feedback plays more important role in glial-glial and glial-neuronal interactions and might be a novel promising molecular target for treatment of CPSP in clinic.

Intervertebral Foramen Injection of Ozone Relieves Mechanical Allodynia and Enhances Analgesic Effect of Gabapentin in Animal Model of Neuropathic Pain.

BACKGROUND: In a 5-year follow-up study in a hospital in southern China, it was shown that intervertebral foramen (IVF) injection of ozone at the involved segmental levels could significantly alleviate paroxysmal spontaneous pain and mechanical allodynia in patients with chronic, intractable postherpetic neuralgia (PHN) and improve the quality of life. However, so far no proof-of-concept studies in animals have been available. OBJECTIVE: This study was designed to investigate whether IVF ozone has an analgesic effect on animal models of neuropathic and inflammatory pain. STUDY DESIGN: Experimental trial in rats. SETTING: Institute for Biomedical Sciences of Pain. METHODS: By IVF injection, a volume of 50 µl containing 30 µg/mL ozone-oxygen mixture or 50 µl air was carried out on male Sprague-Dawley rats of naïve, inflammatory pain states produced by injections of either bee venom or complete Freud's adjuvant, and neuropathic pain state produced by spared nerve injury, respectively. The effects of IVF ozone on pain-related behaviors were evaluated for 2 weeks or one month. Then combined use of gabapentin (100 mg/1 kg body weight) with IVF ozone was evaluated in rats with neuropathic pain by intraperitoneal administration 5 days after the ozone treatment. Finally, the analgesic effects of another 4 drugs, AMD3100 (a CXCR4 antagonist), A-803467 (a selective Nav1.8 blocker), rapamycin (the mTOR inhibitor), and MGCD0103 (a selective histone deacetylase inhibitor) were evaluated for long term through IVF injection, respectively. RESULTS: (1) IVF injection of ozone at L4-5 was only effective in suppression of mechanical allodynia in rats with neuropathic pain but not with inflammatory pain; (2) the analgesic effects of IVF ozone lasted much longer (> 14 days) than other selective molecular target drugs (< 48 hours) inhibiting or antagonizing at Nav1.8 (A-803467), CXCR4 (AMD3100), mTOR (rapamycin), and histone deacetylase (MGCD0103); (3) combined use of systemic gabapentin and IVF ozone produced a synergistic analgesic effect in rats with neuropathic pain. LIMITATIONS: Evaluation of the possible analgesic effects of the intraplantar injection of ozone was not performed. CONCLUSIONS: In the present study, we provided a line of evidence for the first time that IVF injection of ozone selectively relieved neuropathic pain but not inflammatory pain, and enhanced the analgesic effect of gabapentin. KEY WORDS: Chronic pain, neuropathic pain, inflammatory pain, ozone therapy, interventional therapy, gabapentin, spared nerve injury, bee venom, complete Freud's adjuvant.

Ethanol Increases Mechanical Pain Sensitivity in Rats via Activation of GABAA Receptors in Medial Prefrontal Cortex.

Ethanol is widely known for its ability to cause dramatic changes in emotion, social cognition, and behavior following systemic administration in humans. Human neuroimaging studies suggest that alcohol dependence and chronic pain may share common mechanisms through amygdala-medial prefrontal cortex (mPFC) interactions. However, whether acute administration of ethanol in the mPFC can modulate pain perception is unknown. Here we showed that bilateral microinjections of ethanol into the prelimbic and infralimbic areas of the mPFC lowered the bilateral mechanical pain threshold for 48 h without influencing thermal pain sensitivity in adult rats. However, bilateral microinjections of artificial cerebrospinal fluid into the mPFC or bilateral microinjections of ethanol into the dorsolateral PFC (also termed as motor cortex area 1 in Paxinos and Watson's atlas of The Rat Brain. Elsevier Academic Press, Amsterdam, 2005) failed to do so, suggesting regional selectivity of the effects of ethanol. Moreover, bilateral microinjections of ethanol did not change the expression of either pro-apoptotic (caspase-3 and Bax) or anti-apoptotic (Bcl-2) proteins, suggesting that the dose was safe and validating the method used in the current study. To determine whether γ-aminobutyric acid A (GABAA) receptors are involved in mediating the ethanol effects, muscimol, a selective GABAA receptor agonist, or bicuculline, a selective GABAA receptor antagonist, was administered alone or co-administered with ethanol through the same route into the bilateral mPFC. The results showed that muscimol mimicked the effects of ethanol while bicuculline completely reversed the effects of ethanol and muscimol. In conclusion, ethanol increases mechanical pain sensitivity through activation of GABAA receptors in the mPFC of rats.

Melittin, the Major Pain-Producing Substance of Bee Venom.

Melittin is a basic 26-amino-acid polypeptide that constitutes 40-60% of dry honeybee (Apis mellifera) venom. Although much is known about its strong surface activity on lipid membranes, less is known about its pain-producing effects in the nervous system. In this review, we provide lines of accumulating evidence to support the hypothesis that melittin is the major pain-producing substance of bee venom. At the psychophysical and behavioral levels, subcutaneous injection of melittin causes tonic pain sensation and pain-related behaviors in both humans and animals. At the cellular level, melittin activates primary nociceptor cells through direct and indirect effects. On one hand, melittin can selectively open thermal nociceptor transient receptor potential vanilloid receptor channels via phospholipase A2-lipoxygenase/cyclooxygenase metabolites, leading to depolarization of primary nociceptor cells. On the other hand, algogens and inflammatory/pro-inflammatory mediators released from the tissue matrix by melittin's pore-forming effects can activate primary nociceptor cells through both ligand-gated receptor channels and the G-protein-coupled receptor-mediated opening of transient receptor potential canonical channels. Moreover, subcutaneous melittin up-regulates Nav1.8 and Nav1.9 subunits, resulting in the enhancement of tetrodotoxin-resistant Na(+) currents and the generation of long-term action potential firing. These nociceptive responses in the periphery finally activate and sensitize the spinal dorsal horn pain-signaling neurons, resulting in spontaneous nociceptive paw flinches and pain hypersensitivity to thermal and mechanical stimuli. Taken together, it is concluded that melittin is the major pain-producing substance of bee venom, by which peripheral persistent pain and hyperalgesia (or allodynia), primary nociceptive neuronal sensitization, and CNS synaptic plasticity (or metaplasticity) can be readily induced and the molecular and cellular mechanisms underlying naturally-occurring venomous biotoxins can be experimentally unraveled.

Involvement of Rac1 signalling pathway in the development and maintenance of acute inflammatory pain induced by bee venom injection.

BACKGROUND AND PURPOSE: The Rho GTPase, Rac1, is involved in the pathogenesis of neuropathic pain induced by malformation of dendritic spines in the spinal dorsal horn (sDH) neurons. In the present study, the contribution of spinal Rac1 to peripheral inflammatory pain was studied. EXPERIMENTAL APPROACH: Effects of s.c. bee venom (BV) injection on cellular localization of Rac1 in the rat sDH was determined with double labelling immunofluorescence. Activation of Rac1 and its downstream effector p21-activated kinase (PAK), ERKs and p38 MAPK in inflammatory pain states was evaluated with a pull-down assay and Western blotting. The preventive and therapeutic analgesic effects of intrathecal administration of NSC23766, a selective inhibitor of Rac1, on BV-induced spontaneous nociception and pain hypersensitivity were investigated. KEY RESULTS: Rac1 labelling was mainly localized within neurons in both the superficial and deep layers of the sDH in rats of naïve, vehicle-treated and inflamed (BV injected) groups. GTP-Rac1-PAK and ERKs/p38 were activated following s.c. BV injection. Post-treatment with intrathecal NSC23766 significantly inhibited GTP-Rac1 activity and phosphorylation of Rac1-PAK, ERKs and p38 MAPK in the sDH. Both pre-treatment and post-treatment with intrathecal NSC23766 dose-dependently attenuated the paw flinches, primary thermal and mechanical hyperalgesia and the mirror-image thermal hyperalgesia induced by BV injection, but without affecting the baseline pain sensitivity and motor coordination. CONCLUSIONS AND IMPLICATIONS: The spinal GTP-Rac1-PAK-ERK/p38MAPK signalling pathway is involved in both the development and maintenance of peripheral inflammatory pain and can be used as a potential molecular target for developing a novel therapeutic strategy for clinical pain.

Nuclear multidrug-resistance related protein 1 contributes to multidrug-resistance of mucoepidermoid carcinoma mainly via regulating multidrug-resistance protein 1: a human mucoepidermoid carcinoma cells model and Spearman's rank correlation analysis.

BACKGROUND: Multidrug resistance-related protein 1 (MRP1/ABCC1) and multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1) are both membrane-bound drug transporters. In contrast to MDR1, MRP1 also transports glutathione (GSH) and drugs conjugated to GSH. Due to its extraordinary transport properties, MRP1/ABCC1 contributes to several physiological functions and pathophysiological incidents. We previously found that nuclear translocation of MRP1 contributes to multidrug-resistance (MDR) of mucoepidermoid carcinoma (MEC). The present study investigated how MRP1 contributes to MDR in the nuclei of MEC cells. METHODS: Western blot and RT-PCR was carried out to investigate the change of multidrug-resistance protein 1 (MDR1) in MC3/5FU cells after MRP1 was downregulated through RNA interference (RNAi). Immunohistochemistry (IHC) staining of 127 cases of MEC tissues was scored with the expression index (EI). The EI of MDR1 and MRP1 (or nuclear MRP1) was analyzed with Spearman's rank correlation analysis. Using multiple tumor tissue assays, the location of MRP1 in other tissues was checked by HIC. Luciferase reporter assays of MDR1 promoter was carried out to check the connection between MRP1 and MDR1 promoter. RESULTS: MRP1 downregulation led to a decreased MDR1 expression in MC3/5FU cells which was caused by decreased activity of MDR1 promoter. IHC study of 127 cases of MEC tissues demonstrated a strong positive correlation between nuclear MRP1 expression and MDR1 expression. Furthermore, IHC study of multiple tumor tissue array sections showed that although nuclear MRP1 widely existed in MEC tissues, it was not found in normal tissues or other tumor tissues. CONCLUSIONS: Our findings indicate that nuclear MRP1 contributes to MDR mainly through regulating MDR1 expression in MEC. And the unique location of MRP1 made it an available target in identifying MEC from other tumors.

Evaluation of fixation of expandable implants in the mandibles of ovariectomized sheep.

PURPOSE: This study aimed to investigate the effects of an expandable implant (EI) in ovariectomized sheep. METHODS: The EI and taper implant (control group) were produced and placed in mandibles of ovariectomized sheep. Twelve weeks after implantation, resonance frequency analysis, biomechanical tests, histomorphometry, and micro-computed tomography were applied to detect the osseointegration in the 2 groups. RESULTS: The implant stability quotient values, maximal pullout forces, and bone-implant contact (BIC) were 60.3 ± 7.9, 511.0 ± 18.7 N, and 53.14% ± 4.56%, respectively, in the EI group and 58.3 ± 8.9, 394.5 ± 54.5 N, and 46.85% ± 5.04%, respectively, in the control group. There was no significant difference between the 2 groups in implant stability quotient values (P > .05); however, in the EI group the maximal pullout force and BIC were increased significantly (P < .05 and P < .01, respectively). Micro-computed tomography analysis showed that the bone volume/total volume ratio and trabecular number increased significantly (P < .01) and trabecular separation decreased significantly (P < .05) in the EI group. CONCLUSIONS: EI could improve osseointegration in osteoporosis after 12 weeks of implantation by increasing BIC around the implant and by supplying an extra osseointegration surface.

Evaluation of the bactericidal effect of Nd:YAG, Er:YAG, Er,Cr:YSGG laser radiation, and antimicrobial photodynamic therapy (aPDT) in experimentally infected root canals.

BACKGROUND AND OBJECTIVE: In recent years, various laser systems have been introduced into the field of laser-assisted endodontic therapy. The aim of this study was to evaluate the bactericidal effect of Nd:YAG, Er:YAG, Er,Cr:YSGG laser radiation, and antimicrobial photodynamic therapy (aPDT) in experimentally infected root canals compared with standard endodontic treatment of 5.25% sodium hypochlorite (NaClO) irrigation. MATERIALS AND METHODS: Two hundred and twenty infected root canals from extracted human teeth (contaminated with Enterococcus faecalis ATCC 4083 for 4 weeks) were randomly divided into five experimental groups (Nd:YAG, Er:YAG + 5.25% NaClO + 0.9% normal saline + distilled water (Er:YAG/NaClO/NS/DW), Er:YAG + 0.9% normal saline + distilled water (Er:YAG/NS/DW), Er,Cr:YSGG, and aPDT) and two control groups (5.25% NaClO as positive control and 0.9% normal saline (NS) as negative control). The numbers of bacteria on the surface of root canal walls and at different depths inside dentinal tubules before and after treatment were analyzed by means of one-way analysis of variance (one-way ANOVA). The morphology of bacterial cells before and after treatment was examined by scanning electron microscopy (SEM). RESULTS: After treatment, the bacterial reductions in the experimental groups and the positive control group were significantly greater than that of the negative control group (P < 0.001). However, only Er:YAG/NaClO/NS/DW group showed no bacterial growth (the bacterial reduction reached up to 100%) on the surface of root canal walls or at 100/200 µm inside the dentinal tubules. CONCLUSIONS: All the laser radiation protocols tested, especially Er:YAG/NaClO/NS/DW, have effective bactericidal effect in experimentally infected root canals. Er:YAG/NaClO/NS/DW seems to be an ideal protocol for root canal disinfection during endodontic therapy.

Effects of a non-selective TRPC channel blocker, SKF-96365, on melittin-induced spontaneous persistent nociception and inflammatory pain hypersensitivity.

OBJECTIVE: Melittin is the main peptide in bee venom and causes both persistent spontaneous nociception and pain hypersensitivity. Our recent studies indicated that both transient receptor potential (TRP) vanilloid receptor 1 (TRPV1) and canonical TRPs (TRPCs) are involved in mediating the melittin-induced activation of different subpopulations of primary nociceptive cells. Here, we further determined whether TRPC channels are involved in melittin-induced inflammatory nociceptive responses in behavioral assays. METHODS: The anti-nociceptive and anti-hyperalgesic effects of localized peripheral administration of three doses of the non-selective TRPC antagonist, SKF-96365 (1-{ß-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl}-1H-imidazole hydrochloride), were evaluated in melittin tests. Pain-related behaviors were rated by counting the number of paw flinches, and measuring paw withdrawal thermal latency (s) and paw withdrawl mechanical threshold (g), over a 1-h time-course. RESULTS: Localized peripheral SKF-96365 given before melittin prevented, and given after melittin significantly suppressed, the melittin-evoked persistent spontaneous nociception. Pre-blockade and post-suppression of activation of primary nociceptive activity resulted in decreased hypersensitivity to both thermal and mechanical stimuli applied to the primary injury site of the ipsilateral hindpaw, despite dose-effect differences between thermal and mechanical hyperalgesia. However, local administration of SKF-96365 into the contralateral hindpaw had no significant effect on any pain-associated behaviors. In addition, SKF-96365 had no effect on baseline threshold for either thermal or mechanical sensitivity under normal conditions. CONCLUSION: Besides TRPV1, SKF-96365-sensitive TRPC channels might also be involved in the pathophysiological processing of melittin-induced inflammatory pain and hypersensitivity. Therapeutically, SKF-96365 is equally effective in preventing primary thermal and mechanical hyperalgesia as well as persistent spontaneous nociception. However, this drug is likely to be more effective in the relief of thermal hyperalgesia than mechanical hyperalgesia when applied 5 min after establishment of primary afferent activation.

Effects of lateral cortical anchorage on the primary stability of implants subjected to controlled loads: an in vitro study.

Our aim was to evaluate the effects of lateral cortical anchorage on the primary stability of implants subjected to immediate loading. Implants were placed into bovine bones with monocortical anchorage (implant placed through the cortical bone of the crest) and bicortical anchorage (the crest cortical bone plus one cortical bone on the lateral side). Loads of 25N and 50N were applied to the implants in different cycles. The implant stability quotient (ISQ) was measured before and after the cyclic loadings. Under 25N load there was no difference in ISQ between 1800 cyclic loading and preloading, but the values decreased significantly after 3600 cyclic loading in both groups (p<0.05). Under a 50N load the ISQ value after 1800 and 3600 cyclic loading decreased in the monocortical group (p<0.05), but there was no difference between 1800 cyclic loading and preloading in the bicortical group, and the ISQ in the bicortical group was higher than in the monocortical group after 1800 cyclic loading (p<0.05). Our results suggest that the stability of implants with bicortical anchorage decreased more slowly under higher loads.

Nuclear translocation of MRP1 contributes to multidrug resistance of mucoepidermoid carcinoma.

Multidrug resistance-related protein 1 (MRP1 or ABCC1), a membrane-bound energy-dependent efflux transporter, is overexpressed in several kinds of multidrug-resistant cell lines and related to multidrug-resistance (MDR) of various cancers. In this study, we investigated whether MRP1 was involved in the chemoresistance of mucoepidermoid carcinoma (MEC). We demonstrated that down-regulation of MRP1 in MC3/5FU, a drug-resistant MEC cell line, by RNA interference increased the drug sensitivity of the cells to 5-fluorouracil, doxorubicin, pharmorubicin, bleomycin-A5, cis-platinum and taxol. However, no significant quantitative difference of MRP1 mRNA and protein expression was found between MC3/5FU cells and its parental cell line (MC3) as determined by RT-PCR and Western blot. Interestingly, MRP1 was translocated from the cytoplasmic membrane of the MC3 cells to the nuclei of MC3/5FU cells as revealed by indirect immunofluorescence staining. Furthermore, MRP1 down-regulation mainly decreased the nuclear expression of MRP1 rather than the cytoplasmic membrane expression. Our results suggested that MRP1 was involved in the chemoresistance of MEC and MRP1 may confer drug-resistance by a mechanism associated with its nuclear translocation.

Antisense-mediated knockdown of Na(V)1.8, but not Na(V)1.9, generates inhibitory effects on complete Freund's adjuvant-induced inflammatory pain in rat.

Tetrodotoxin-resistant (TTX-R) sodium channels Na(V)1.8 and Na(V)1.9 in sensory neurons were known as key pain modulators. Comparing with the widely reported Na(V)1.8, roles of Na(V)1.9 on inflammatory pain are poorly studied by antisense-induced specific gene knockdown. Here, we used molecular, electrophysiological and behavioral methods to examine the effects of antisense oligodeoxynucleotide (AS ODN) targeting Na(V)1.8 and Na(V)1.9 on inflammatory pain. Following complete Freund's adjuvant (CFA) inflammation treatment, Na(V)1.8 and Na(V)1.9 in rat dorsal root ganglion (DRG) up-regulated mRNA and protein expressions and increased sodium current densities. Immunohistochemical data demonstrated that Na(V)1.8 mainly localized in medium and small-sized DRG neurons, whereas Na(V)1.9 only expressed in small-sized DRG neurons. Intrathecal (i.t.) delivery of AS ODN was used to down-regulate Na(V)1.8 or Na(V)1.9 expressions confirmed by immunohistochemistry and western blot. Unexpectedly, behavioral tests showed that only Na(V)1.8 AS ODN, but not Na(V)1.9 AS ODN could reverse CFA-induced heat and mechanical hypersensitivity. Our data indicated that TTX-R sodium channels Na(V)1.8 and Na(V)1.9 in primary sensory neurons played distinct roles in CFA-induced inflammatory pain and suggested that antisense oligodeoxynucleotide-mediated blocking of key pain modulator might point toward a potential treatment strategy against certain types of inflammatory pain.

The biomechanical analysis of simulating implants in function under osteoporotic jawbone by comparing cylindrical, apical tapered, neck tapered, and expandable type implants: a 3-dimensional finite element analysis.

PURPOSE: This study compared the biomechanical behaviors of 4 implants in osteoporosis by 3-dimensional finite element analyses. MATERIALS AND METHODS: Finite element models (FEM) of posterior mandible segments with a cylindrical threaded implant, an apical tapered implant, a neck tapered implant (NTI), and an apical expandable implant were created. Bone segments with normal and osteoporotic biomechanical properties were used. Forces of 100 and 30 N were applied along the implant in axial and buccolingual (BL) directions, respectively. Maximum equivalent stresses in the jaw bone and maximum displacement in the implant-abutment complex were evaluated. RESULTS: In osteoporosis, compared with the cylindrical threaded implant, maximum equivalent stress in cortical bones with the apical tapered implant decreased by 10.1% and 6.57% under axial and BL loads, respectively. With the NTI, those values decreased by 10.72% and 7.87%. With the apical expandable implant, those values decreased by 11.3% and 9.60%. In cancellous bones, the maximum equivalent stress with the NTI decreased by 3.56% under a BL load. Maximum displacement in the implant-abutment complex decreased by 17.1% and 9.41% with an apical tapered implant under axial and BL loads, respectively. With the NTI, those decreased by 21.8% and 17.4%. Values in normal bone indicated better stress distributions and less displacement than those in osteoporotic bone. CONCLUSION: Stress distribution in the jaw bone and implant stability in osteoporotic bone were more sensitive to implant designs than those in normal bone. In osteoporotic bone, the expandable implant and the NTI showed better stress distribution, and tapered implants showed better stabilities.

Prevotella intermedia stimulates tissue-type plasminogen activator and plasminogen activator inhibitor-2 expression via multiple signaling pathways in human periodontal ligament cells.

Prevotella intermedia is an important periodontal pathogen that induces various inflammatory and immune responses. In this study, we investigated the effects of P. intermedia on the plasminogen system in human periodontal ligament (hPDL) cells and explored the signaling pathways involved. Using semi-quantitative reverse transcription (RT)-PCR and quantitative real-time RT-qPCR, we demonstrated that P. intermedia challenge increased tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)-2 expression in a concentration- and time-dependent manner, but exerted no influence on urokinase-type plasminogen activator and PAI-1mRNA expression in hPDL cells. Prevotella intermedia stimulation also enhanced tPA protein secretion as confirmed by enzyme-linked immunosorbent assay. Western blot results revealed that P. intermedia treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase (p38). ERK, JNK and protein kinase C inhibitors significantly attenuated the P. intermedia-induced tPA and PAI-2 expression. Furthermore, p38 and phosphatidylinositol 3-kinase inhibitors markedly decreased PAI-2 expression, whereas they showed no or little inhibition on tPA expression. In contrast, inhibition of protein kinase A greatly enhanced the upregulatory effect of P. intermedia on tPA and PAI-2 expression. Our results suggest that P. intermedia may contribute to periodontal tissue destruction by upregulating tPA and PAI-2 expression in hPDL cells via multiple signaling pathways.

Baicalin induces human mucoepidermoid carcinoma Mc3 cells apoptosis in vitro and in vivo.

Mucoepidermoid carcinoma (MEC) is the most common malignant tumor in salivary glands and high-grade MEC in particular demonstrates little response to chemotherapy which has been used largely for palliative treatment of metastatic disease. Baicalin, one of the main active compounds of Scutellaria baicalensis, possesses anti-inflammatory, antioxidant and antitumor properties. In the present study, we investigated the growth inhibiting and apoptosis-inducing effects of baicalin on a highly metastatic human mucoepidermoid carcinoma cell line Mc3 for the first time. Baicalin exerted dose- and time-dependent antiproliferative potential against Mc3 cells as assessed by MTT assay. Baicalin treatment of Mc3 cells resulted in an accumulation of cells at the G0/G1 and G2/M phase with a concomitant decrease in cells processing to S phase as assessed by flow cytometry. Furthermore, baicalin induced apoptosis of Mc3 cells as determined by annexin V binding and PI dual staining, DNA fragmentation, nuclear condensation and in vivo tumor inhabitation. Rhodamine 123 assay indicated that baicalin caused cytotoxicity and induced apoptosis through decreasing the mitochondrial membrane potential in Mc3 cells. Our results suggest that baicalin seems to be very attractive as a new anticancer drug and a potential chemotherapeutic agent against human high-grade mucoepidermoid carcinoma.

[PTEN tumor suppressor gene combined with doxycycline inhibites telomerase activity in human mucoepidermoid carcinoma cell line].

OBJECTIVE: To investigate the effect of PTEN tumor suppressor gene combined with doxycycline on telomerase activity in human mucoepidermoid carcinoma cell line. METHODS: The wild-type PTEN tumor suppressor gene or empty vector was introduced into mucoepidermoid carcinoma cell line in vitro, then the cancer cells were treated with doxycycline. Cancer cell survival was determined by MTT assay. Telomerase activity was determined using telomerase repeat amplification protocol-enzyme-linked immunosorbent assay (TRAP-ELISA). RESULTS: Compared to the control cells, cancer cells transfected with the wild-type PTEN gene showed growth inhibition and increased sensitivity to doxycycyline, and the ratio of augment of drug sensitivity was 1.65-4.75. The telomerase activity in cancer cells treared with PTEN gene transfection or doxycycline alone decreased, however, telomerase activity in combined group decreased more remarkably. CONCLUSION: PTEN gene in combination with doxycycline has significant inhibitory effect on telomerase activity in cancer cells.

Prevotella intermedia upregulates MMP-1 and MMP-8 expression in human periodontal ligament cells.

Prevotella intermedia, a major periodontal pathogen, plays important roles in the initiation and development of periodontitis by stimulating the release of proinflammatory cytokines, proteinases and matrix metalloproteinases (MMPs). Our previous study demonstrated that P. intermedia induced MMP-9 expression in human periodontal ligament (hPDL) cells. In this study, we examined the effects of P. intermedia on other MMPs' expression. Semi-quantitative reverse transcriptase (RT)-PCR analysis revealed that P. intermedia ATCC 25611 supernatant increased MMP-1 and MMP-8 mRNA expression in a concentration- and time-dependent manner. Enzyme-linked immunosorbent assay and Western blot results confirmed the RT-PCR results at the protein level. Cyclooxygenase inhibitor indomethacin significantly attenuated the upregulatory effects of P. intermedia on MMP-1 and MMP-8 expression. Extracellular signal-related kinase inhibitor PD98059 and c-Jun N-terminal kinase inhibitor SP600125 considerably decreased the upregulated level of MMP-1, whereas p38 inhibitor SB203580 markedly inhibited MMP-8 expression, suggesting that prostaglandin E(2) and mitogen-activated protein kinase signaling pathways are involved in P. intermedia-induced MMP-1 and MMP-8 upregulation. Our results indicate that P. intermedia might contribute to periodontal connective tissue and bone matrix destruction through upregulating MMP production.

Mitogen-activated protein kinases and phosphatidylinositol 3-kinase are involved in Prevotella intermedia-induced proinflammatory cytokines expression in human periodontal ligament cells.

Chronic periodontitis is an inflammatory disease affecting periodontal connective tissues and alveolar bone. Proinflammatory mediators induced by periodontal pathogens play vital roles in the initiation and progression of the disease. In this study, we examined whether Prevotella intermedia induces proinflammatory cytokines expression in human periodontal ligament cells (hPDLs). The mRNA expression and protein production were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay (ELISA) respectively. P. intermedia treatment dose- and time-dependently increased IL-6, IL-8 and M-CSF, but not IL-1beta and TNF-alpha mRNA expression and protein secretion. Preincubation of hPDLs with extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 kinase and phosphatidylinositol 3-kinase (PI3K) inhibitors PD98059, SP600125, SB203580 and LY294002 resulted in significant reduction in P. intermedia-induced IL-6, IL-8 and M-CSF expression. Blocking the synthesis of prostaglandin E(2) (PGE(2)) by indomethacin also abolished the stimulatory effects of P. intermedia on cytokines expression. Our results indicate that P. intermedia induces proinflammatory cytokines through MAPKs and PI3K signaling pathways, and PGE(2) is involved in the P. intermedia-induced proinflammatory cytokines upregulation.