Effectiveness of Stitch With Pledget to Prevent Prolonged Air Leak in Thoracoscopic Lung Resection.

INTRODUCTION: We previously demonstrated the usefulness of combining stitching with covering to seal alveolar air leaks in an animal model. This study aimed to clarify the effectiveness and feasibility of this sealing method in the clinical setting. METHODS: Data of 493 patients who underwent thoracoscopic anatomical resection between 2013 and 2020 for lung cancer were retrospectively reviewed. Prolonged air leak was defined as chest drain placement lasting 5 d or longer due to air leak. Until July 2017 (early study period), we covered air leaks using mesh. However, for sealing (late study period), we additionally stitched leaks with pledget in patients at high risk of prolonged air leak. The pneumostasis procedure, intraoperative confirmation test of pneumostasis, and chest tube management were uniform during both periods. RESULTS: The incidence of prolonged air leak was significantly lower in the late than in the early period (3.6% versus 12.5%), whereas pulmonary emphysema was more severe in the late period compared to the early period. Intraoperative failure of sealing air leaks was significantly reduced in the late period than in the early period. In both univariate and propensity score matching analysis, the study period was a significant predictor of prolonged air leak. CONCLUSIONS: The combination of stitching and covering with mesh may contribute to reducing prolonged air leak incidence in patients undergoing thoracoscopic anatomical lung resection for lung cancer.

Cilostazol treats transient heart failure caused by ATP1A3 variant-associated polymicrogyria.

BACKGROUND: Some patients with ATP1A3 variant-associated polymicrogyria have recurrent transient heart failure. However, effective treatment for the transient cardiac condition remains to be elucidated. CASE REPORT: The patient started experiencing focal motor onset seizures in 12 h after birth, revealing bilateral diffuse polymicrogyria. The patient also experienced transient bradycardia (sinus bradycardia) attacks from 15 days old. Echocardiography revealed a reduced ejection fraction; however, no obvious electrocorticogram or electroencephalogram abnormalities were observed during the attacks. Initially, the attacks occurred in clusters daily. They later decreased in frequency, occurring at monthly intervals. Repeated episodes of transient bradycardia attacks and polymicrogyria indicated possible ATP1A3 gene abnormality and genetic testing revealed a novel heterozygous ATP1A3 variant (NM_152296: exon22:c.2977_2982del:p.(Glu993_Ile994del)), which was not found in the patient's parents. Cilostazol was administered at 3 months old for recurrent transient bradycardia attacks. Cilostazol significantly shortened the duration of bradycardia episodes and prolonged the interval between attacks. Cilostazol also effectively treats transient symptomatic bradycardia. CONCLUSION: Cilostazol could be a treatment option for recurrent transient bradycardia attacks associated with ATP1A3 gene abnormalities and polymicrogyria.

Laparoscopic-assisted thoracoscopic repair of latent traumatic diaphragmatic hernia: A case report.

Surgical approaches for traumatic diaphragmatic hernia include transabdominal, transthoracic, and thoracoabdominal. Selection of the optimal approach depends on the timing and organ damage, often minimally invasive approaches with laparoscopy or thoracoscopy are performed. A 47-year-old man with blunt chest trauma was diagnosed with left traumatic diaphragmatic hernia 1 month after the trauma. The prolapsed omentum was detached from the chest wall and around the hernia orifice and returned to the abdominal cavity by coordinated thoracoscopic and laparoscopic manipulations. The 4 × 2 cm herniation in the diaphragm was sutured closed from the thoracic side while preventing re-prolapse of the omentum and abdominal organs from the abdominal side. A combined thoracoscopic and laparoscopic approach can be effective in confirming organ damage, repositioning of prolapsed organs, and safe repair of the diaphragm in latent traumatic diaphragmatic hernia.

Allogenic multilayered fibroblast sheets promote anastomotic site healing in a rat model of esophageal reconstruction.

OBJECTIVE: Anastomotic leakage is a common and severe complication of esophageal reconstruction. Accordingly, there is a clinical need for novel methods to prevent it. We developed multilayered, growth factor-secreting fibroblast sheets that promote wound healing and angiogenesis. The present study aimed to assess the utility of allogenic multilayered fibroblast sheets in preventing esophageal anastomotic leakage in a rat model of esophageal reconstruction. METHODS: Allogenic multilayered fibroblast sheets prepared from oral mucosal tissues were implanted at esophageal anastomotic sites. RESULTS: The allogenic multilayered fibroblast sheet group had significantly higher burst pressure and collagen deposition compared to a control group five days postoperatively. The expression levels of collagen type I and III mRNAs around esophageal suture sites were higher in the allogenic multilayered fibroblast sheet group compared to the control group on postoperative days 0, 3, and 5. There was a trend toward lower anastomotic leakage and lower abscess scores in the allogenic multilayered fibroblast sheet group compared to the control group; however, these differences did not reach statistical significance. Allogenic multilayered fibroblast sheets completely disappeared at ten days after implantation. Further, no inflammation was observed at suture sites with implanted allogenic multilayered fibroblast sheets at five days after surgery. CONCLUSION: Allogenic multilayered fibroblast sheets may represent a promising method of preventing esophageal anastomotic leakage.

Postoperative changes in a bronchial stump following covering with free fat tissue in a rat model.

OBJECTIVES: Covering the bronchial stump with free fat tissue has been used as minimally invasive prophylaxis against bronchial stump fistulas; however, postoperative changes in the bronchial stump have not been well validated. Our goal was to examine changes in the bronchial stump in response to covering with free fat tissue in a rat model. METHODS: A left pneumonectomy was performed on 16 Wistar/ST rats, 12 of which had a bronchial stump covered with free subcutaneous fat tissue. Four rats that underwent a left pneumonectomy alone were sacrificed on postoperative day 7, and the 12 rats whose bronchial stumps were additionally covered with fat tissue were sacrificed on postoperative days 7, 14 and 56. Macroscopic and histological changes and pressure resistance of the bronchial stumps due to coverage with free fat tissue were examined. RESULTS: None of the rats showed macroscopic infection or necrosis in the thoracic cavity at the time of the rethoracotomy. The normal bronchial stumps remained mostly exposed, whereas the bronchial stumps covered with fat tissue were well-coated with tissue mass. Histologically, fibrous connective tissue containing microvessels gradually formed around the bronchial stump covered with fat tissue, and some of the tissue masses still had normal fat structures 56 days postoperatively. Covering with fat tissue significantly increased the pressure resistance of the bronchial stump 7 days postoperatively and further increased with time. CONCLUSIONS: Covering the bronchial stump with free fat tissue formed fibrous connective tissue around the bronchial stump and reinforced its closure.

Late relapse of anti-N-methyl-d-aspartate receptor encephalitis with amusia and transiently reduced uptake in 123I-iomazenil single-photon emission computed tomography.

INTRODUCTION: Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis has a high relapse rate at approximately 10-20%. Most relapses occur within 2 years from onset, and 5 years after onset is rare. We report a case of anti-NMDAR encephalitis relapse with amusia 10 years after the initial encephalitis and discuss the usefulness of 123I-iomazenil single-photon emission computerized tomography (IMZ-SPECT) for its diagnosis. CASE: A 13-year-old left-handed girl presented with a depressed level of consciousness and focal to bilateral tonic-clonic seizures. Cerebrospinal fluid (CSF) analysis showed a mildly increased white blood cell count, elevated neopterin levels, and positive oligoclonal bands. Brain MRI was normal. IMZ-SPECT revealed reduced uptake in the right frontoparietal region. She received intravenous pulse methylprednisolone (IVMP) and high-dose intravenous immunoglobulin for autoimmune encephalitis; her symptoms resolved without neurological deficits. At 23 years old, she had mild right-sided numbness, dysarthria, amusia, and tonic-clonic seizures. Although the CSF analysis and brain MRI were normal, IMZ-SPECT revealed reduced uptake, indicating a relapse of encephalitis. IVMP administration resolved the symptoms. After discharge, the initial and relapse CSF analysis revealed anti-NMDAR antibodies. CONCLUSION: An anti-NMDAR encephalitis relapse 10 years after onset has never been reported. IMZ-SPECT may help in the diagnosis of anti-NMDAR encephalitis.

A case of bilateral limbic and recurrent unilateral cortical encephalitis with anti-myelin oligodendrocyte glycoprotein antibody positivity.

BACKGROUND: Anti-myelin oligodendrocyte glycoprotein (MOG) antibody can be detected not only in acute disseminated encephalomyelitis or optic neuritis but also in limbic or cortical encephalitis. However, no previous reports have demonstrated a relapsing case of these two types of encephalitis. CASE REPORT: An 11-year-old girl presented with fever, headache, abnormal behavior, focal impaired awareness seizures (FIAS) on the left side, and MRI hyperintensities in the bilateral amygdala, hippocampus, and right posterior temporal cortex. The symptoms were alleviated with two courses of intravenous methylprednisolone (IVMP) and one course of immunoglobulin. At 16 years of age, the patient returned with left-sided headache and MRI hyperintensities in the left temporal, parietal, and insular cortices, which improved after 3 courses of IVMP. Oral prednisolone (PSL) was tapered over 6 months, when FIAS reappeared on the right side of the body. MRI showed recurrence in the same regions as in the second episode. She received 3 courses of IVMP, followed by gradually tapered PSL without relapse for 1.5 year. Anti-MOG antibodies were positive in both serum and the cerebrospinal fluid prior to treatment in all three episodes. CONCLUSION: Our results revealed that anti-MOG antibody-related bilateral limbic and unilateral cortical encephalitis can manifest with a variety of phenotypes over time in the same patient.

Chemotherapy-induced autoimmune-mediated encephalitis during germinoma treatment.

BACKGROUND: Autoimmune mediated encephalitis (AME), which includes autoantibody-associated encephalitis and acute disseminated encephalomyelitis, is a common cause of encephalitis as well as infectious encephalitis in children. AME may be triggered by autoimmune responses to paraneoplastic syndromes and infections. Infectious encephalitis associated with an immunocompromised status caused by anti-cancer chemotherapy is well recognized; however, there have been few reports on the relationship between AME and chemotherapy. CASE REPORT: A ten-year-old previously healthy, developmentally normal girl was diagnosed with a pure germinoma in the suprasellar region. Following 30 days of induction chemotherapy, she developed a depressed level of consciousness with accompanying right hemiplegia, aphasia, and unexplained fever. Cerebrospinal fluid (CSF) analysis revealed positive oligoclonal bands and elevated neopterin levels. Neither atypical cells suggesting tumor exacerbation nor pathogens known to cause encephalitis were identified in the CSF. She was administrated immunosuppressive therapy and her symptoms rapidly improved. No known autoantibodies associated with autoantibody-associated encephalitis were identified in blood or CSF. However, the presence of oligoclonal bands and elevated neopterin levels in the CSF, and the favorable response to immunosuppressive therapy were consistent with an AME diagnosis. Thirteen days after the third course of chemotherapy, the patient developed a depressed level of consciousness again. Due to the recurrence of encephalitis, re-administration of immunosuppressive therapy was performed, which led to improvement in her symptoms. Recurrence of encephalitis has not occurred for 1 year after completion of chemotherapy. CONCLUSION: The chemotherapy-induced abnormal immune response might have triggered the AME.

Distinct dual cortico-cortical networks successfully identified between supplemental and primary motor areas during intracranial EEG for drug-resistant frontal lobe epilepsy.

We present a case of drug-resistant focal motor seizures in which separate cortico-cortical epileptic networks within the supplementary motor area (SMA) proper and primary motor area (PMA) were proven by ictal high-frequency oscillation (HFO) and cortico-cortical evoked potential (CCEP). A 12-year-old girl presented with two types seizures: type A, tonic extension and subsequent clonic movements of the right arm; and type B, tonic and clonic movements of the right leg. MRI was normal and karyotype genetic analysis revealed 46,X,t(X;14)(q13;p12). She underwent placement of chronic subdural electrodes over the left hemisphere. We recorded a total of nine seizures during 10 days of epilepsy monitoring. Type A seizures started from the lower part of the left SMA proper and early spread to the hand motor area of the PMA. Type B seizures started from the upper part of the SMA proper and early spread to the leg motor area of the PMA. CCEPs of both SMA proper and PMA activated two identical routes for evoked potentials correlating with separate pathways. Corticectomy of the left SMA proper and PMA achieved seizure-free without hemiparesis. Within a small homunculus of the SMA proper, separate epileptic networks were proven and validated by seizure semiology, ictal HFO, and CCEP.

HSP22 (HSPB8) positively regulates PGF2α-induced synthesis of interleukin-6 and vascular endothelial growth factor in osteoblasts.

BACKGROUND: Heat shock protein 22 (HSP22) belongs to class I of the small HSP family that displays ubiquitous expression in osteoblasts. We previously demonstrated that prostaglandin F2α (PGF2α), a potent bone remodeling factor, induces the synthesis of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether HSP22 is implicated in the PGF2α-induced synthesis of IL-6 and VEGF and the mechanism of MC3T3-E1 cells. METHODS: MC3T3-E1 cells were transfected with HSP22-siRNA. IL-6 and VEGF release was assessed by ELISA. Phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was detected by Western blotting. RESULTS: The PGF2α-induced release of IL-6 in HSP22 knockdown cells was significantly suppressed compared with that in the control cells. HSP22 knockdown also reduced the VEGF release by PGF2α. Phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was attenuated by HSP22 downregulation. CONCLUSIONS: Our results strongly suggest that HSP22 acts as a positive regulator in the PGF2α-induced synthesis of IL-6 and VEGF in osteoblasts.

Association of HSP22 with mTOR in osteoblasts: regulation of TNF-α-stimulated IL-6 synthesis.

Heat shock protein 22 (HSP22) is ubiquitously expressed in various types of cells including in osteoblasts. We previously reported that tumor necrosis factor (TNF)-α stimulates interleukin (IL)-6 synthesis via p44/p42 MAPK in osteoblast-like MC3T3-E1 cells and that mTOR/p70 S6 kinase (p70 S6K) negatively regulates the IL-6 synthesis. In this study, we investigated the involvement of HSP22 in TNF-α-stimulated-IL-6 synthesis and the underlying mechanism in MC3T3-E1 cells. HSP22 knockdown reduces TNF-α-stimulated release of IL-6. In addition, HSP22 knockdown strengthens TNF-α-induced phosphorylation of p70 S6K but suppresses that of p44/p42 MAPK. HSP22 coimmunoprecipitates with mTOR. HSP22 knockdown increases the basal levels of phosphorylated mTOR. These results strongly suggest that HSP22 interacts with mTOR and regulates TNF-α-induced IL-6 synthesis in osteoblasts.

Treatment of Lateral Tibial Condylar Fractures Using Bioactive, Bioresorbable Forged Composites of Raw Particulate Unsintered Hydroxyapatite/Poly-L-Lactide Screws.

Forged composites of raw particulate unsintered hydroxyapatite/poly-L-lactide (F-u-HA/PLLA) devices possess high mechanical strength, bioactivity, and radio-opacity. The aim of this study was to assess the efficacy of F-u-HA/PLLA screws in the treatment of lateral tibial condylar fractures. From January 2005 to December 2010, a total of 7 patients with displaced closed lateral tibial condylar fractures (Schatzker type II) were treated using F-u-HA/PLLA screws. Open reduction and internal fixation was performed using 2 or 3 F-u-HA/PLLA screws. After surgery, weight bearing was not allowed for 6 weeks. Range of motion exercise was initiated after removal of the plaster splint. Radiographs were evaluated for fracture healing, joint depression, and the radioopacity of F-u-HA/PLLA screws. Clinical outcomes and postoperative complications were also assessed. Average follow-up was 44 months. All fractures were successfully healed. Average values for joint depression were 4.7 mm (range, 2-9 mm) preoperatively, 0.4 mm (range, 0-1 mm) postoperatively, and 0.4 mm (range, 0-1 mm) at final follow-up. Whole shadows of F-u-HA/PLLA screws were observed during the follow-up period. Breakage of screws, osteolysis, and a radiolucent zone around the screws were not observed at final follow-up. Average knee flexion and extension were 134° (range, 110° to 150°) and -1° (range, -10° to 0°), respectively. No patient had wound infection, late aseptic tissue response, or foreign body reaction postoperatively. None of the patients reported pain at final follow-up. These results suggest that F-u-HA/PLLA screws could be an alternative option for the treatment of lateral tibial condylar fractures. [Orthopedics. 2018; 41(3):e365-e368.].

Suppression by HSP90 inhibitors of BMP­4­stimulated osteoprotegerin synthesis in osteoblasts: Attenuation of p70 S6 kinase.

Heat shock protein 90 (HSP90) is an ATP­dependent ubiquitous molecular chaperon which is important in cell homeostasis. The authors previously demonstrated that bone morphogenetic protein (BMP)­4 stimulates osteoprotegerin (OPG) production in osteoblast­like MC3T3­E1 cells, and that p70 S6 kinase positively regulates the OPG synthesis by BMP­4. The present study investigated the involvement of HSP90 in the BMP­4­stimulated OPG synthesis and the mechanism in MC3T3­E1 cells. HSP90 inhibitors, 17­allylamino­17demethoxy­geldanamycin (17­AAG), 17­dimethylamino­ethylamino­17­demethoxy­geldanamycin (17­DMAG) and geldanamycin significantly suppressed the BMP­4­stimulated OPG release. Geldanamycin markedly reduced the BMP­4­induced mRNA expression of OPG. 17­AAG and 17­DMAG significantly attenuated the phosphorylation of p70 S6 kinase induced by BMP­4 without affecting the BMP­4­induced phosphorylation of mothers against decapentaplegic homolog 1/5. The results suggest that HSP90 inhibitors suppress the BMP­4­stimulated OPG synthesis in osteoblasts, and that their suppressive effects are exerted through downregulating p70 S6 kinase.

Heat shock protein 27 (HSPB1) suppresses the PDGF-BB-induced migration of osteoblasts.

Heat shock protein 27 (HSP27/HSPB1), one of the small heat shock proteins, is constitutively expressed in various tissues. HSP27 and its phosphorylation state participate in the regulation of multiple physiological and pathophysiological cell functions. However, the exact roles of HSP27 in osteoblasts remain unclear. In the present study, we investigated the role of HSP27 in the platelet-derived growth factor­BB (PDGF­BB)­stimulated migration of osteoblast-like MC3T3-E1 cells. PDGF-BB by itself barely upregulated the expression of HSP27 protein, but stimulated the phosphorylation of HSP27 in these cells. The PDGF-BB­induced cell migration was significantly downregulated by HSP27 overexpression. The PDGF-BB-induced migrated cell numbers of the wild­type HSP27-overexpressing cells and the phospho­mimic HSP27-overexpressing (3D) cells were less than those of the unphosphorylatable HSP27-overexpressing (3A) cells. PD98059, an inhibitor of MEK1/2, SB203580, an inhibitor of p38 mitogen-activated protein kinase, and SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) reduced the PDGF-BB-induced migration of these cells, whereas Akt inhibitor or rapamycin, an inhibitor of upstream kinase of p70 S6 kinase (mTOR), barely affected the migration. However, the PDGF-BB-induced phosphorylation of p44/p42 MAPΚ, p38 MAPK and SAPK/JNK was not affected by HSP27 overexpression. There were no significant differences in the phosphorylation of p44/p42 MAPΚ, p38 MAP kinase or SAPK/JNK between the 3D cells and the 3A cells. These results strongly suggest that HSP27 functions as a negative regulator in the PDGF-BB-stimulated migration of osteoblasts, and the suppressive effect is amplified by the phosphorylation state of HSP27.

Resveratrol suppresses thyroid hormone­induced osteocalcin synthesis in osteoblasts.

Resveratrol, a polyphenolic compound that is present in grape skins, berries and red wine, may be beneficial for human health through its anti­inflammatory and anti­oxidant effects. It has been previously demonstrated that resveratrol exerts its biological effects primarily via sirtuin 1 (SIRT1) activation. We previously reported that triiodothyronine (T3) induces osteocalcin synthesis in osteoblast­like MC3T3­E1 cells, and that p38 mitogen­activated protein (MAP) kinase mediates the T3­stimulated synthesis of osteocalcin. The present study investigated the effect of resveratrol on T3­induced osteocalcin synthesis and its underlying mechanism in MC3T3­E1 cells. Cultured cells were stimulated with T3, and osteocalcin release from MC3T3­E1 cells was measured by ELISA and phosphorylation of p38 MAP kinase was analyzed by western blotting. Resveratrol significantly suppressed the release of osteocalcin stimulated by T3, and SRT1720, a SIRT1 activator, significantly reduced T3­induced osteocalcin release. The expression level of osteocalcin mRNA stimulated by T3 was significantly attenuated by resveratrol and T3­induced transactivation activity of the thyroid hormone­responsive element was significantly diminished by resveratrol. However, only limited effects of resveratrol on the T3­induced phosphorylation of p38 MAP kinase were observed. The results of the present study demonstrated that resveratrol suppresses T3­stimulated osteocalcin synthesis at a point upstream of transcription in osteoblasts, and that the inhibitory effect of resveratrol is mediated, at least partially, through SIRT1 activation. These results indicate that there may be a novel role for the polyphenol in the modulation of bone metabolism.

HSP90 inhibitors potentiate PGF2α-induced IL-6 synthesis via p38 MAP kinase in osteoblasts.

Heat shock protein 90 (HSP90) that is ubiquitously expressed in various tissues, is recognized to be a major molecular chaperone. We have previously reported that prostaglandin F2α (PGF2α), a potent bone remodeling mediator, stimulates the synthesis of interleukin-6 (IL-6) through p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that Rho-kinase acts at a point upstream of p38 MAP kinase. In the present study, we investigated the involvement of HSP90 in the PGF2α-stimulated IL-6 synthesis and the underlying mechanism in MC3T3-E1 cells. Geldanamycin, an inhibitor of HSP90, significantly amplified both the PGF2α-stimulated IL-6 release and the mRNA expression levels. In addition, other HSP90 inhibitors, 17-allylamino-17demethoxy-geldanamycin (17-AAG) and 17-dimethylamino-ethylamino-17-demethoxy-geldanamycin (17-DMAG) and onalespib, enhanced the PGF2α-stimulated IL-6 release. Geldanamycin, 17-AAG and onalespib markedly strengthened the PGF2α-induced phosphorylation of p38 MAP kinase. Geldanamycin and 17-AAG did not affect the PGF2α-induced phosphorylation of p44/p42 MAP kinase and myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase, and the protein levels of RhoA and Rho-kinase. In addition, HSP90-siRNA enhanced the PGF2α-induced phosphorylation of p38 MAP kinase. Furthermore, SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by geldanamycin, 17-AAG or 17-DMAG of the PGF2α-stimulated IL-6 release. Our results strongly suggest that HSP90 negatively regulates the PGF2α-stimulated IL-6 synthesis in osteoblasts, and that the effect of HSP90 is exerted through regulating p38 MAP kinase activation.

Attenuation of prostaglandin E1­induced osteoprotegerin synthesis in osteoblasts by normoxic HIF inducers.

Mimosine, which is a natural plant amino acid present in the Leucaena genus, is able to induce hypoxia­inducible factors (HIFs). Previous evidence has indicated that HIF regulates angiogenesis­osteogenesis coupling in bone metabolism, and it has previously been reported that mimosine inhibits prostaglandin (PG)F2α­induced osteoprotegerin (OPG) synthesis without affecting interleukin­6 (IL­6) production in osteoblast­like MC3T3­E1 cells. In addition, PGE1 has been demonstrated to induce OPG synthesis via activation of p38 mitogen­activated protein (MAP) kinase and stress­activated protein kinase/c­Jun N­terminal kinase (SAPK/JNK) in these cells, and PGE1 stimulates IL­6 production via the activation of protein kinase A. In the present study, the effects of mimosine on the PGE1­stimulated synthesis of OPG and IL­6 were investigated in osteoblast­like MC3T3­E1 cells. The concentrations of OPG and IL­6 were measured using relevant ELISA kits. OPG mRNA was measured by semi­quantitative reverse transcription polymerase chain reaction. The phosphorylation of p38 MAP kinase and SAPK/JNK was analyzed by western blotting. Mimosine significantly reduced PGE1­induced release of OPG and OPG mRNA expression levels without affecting the release of IL­6. In addition, deferoxamine, which is also a normoxic HIF inducer, significantly inhibited PGE1­induced OPG release and OPG mRNA expression levels; however, it had little effect on IL­6 release. Furthermore, mimosine and deferoxamine failed to affect PGE1­stimulated phosphorylation of p38 MAP kinase or SAPK/JNK. These results strongly suggest that normoxic HIF inducers attenuate PGE1­stimulated OPG synthesis without affecting IL­6 production in osteoblasts.

(-)-Epigallocatechin gallate synergistically potentiates prostaglandin E2-stimulated osteoprotegerin synthesis in osteoblasts.

(-)-Epigallocatechin gallate (EGCG), the most abundant flavonoid in green tea, and chlorogenic acid, the main polyphenol found in coffee, attract significant attention owing to health benefits. We have previously demonstrated that prostaglandin E2 (PGE2) stimulates osteoprotegerin synthesis through the activation of p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of EGCG or chlorogenic acid on the PGE2-stimulated osteoprotegerin synthesis in MC3T3-E1 cells. EGCG significantly amplified the PGE2-induced release. EGCG markedly enhanced the expression levels of osteoprotegerin mRNA induced by PGE2. On the contrary, chlorogenic acid had no effect on the PGE2-stimulated release of osteoprotegerin. EGCG significantly strengthened the PGE2-induced phosphorylation of p38 MAP kinase and SAPK/JNK, whereas chlorogenic acid failed to affect them. BIRB0796 and SP600125, a p38 MAP kinase inhibitor and a SAPK/JNK inhibitor, respectively, markedly reduced the amplification by EGCG of the PGE2-stimulated osteoprotegerin release. These results strongly suggest that EGCG synergistically enhances the PGE2-stimulated osteoprotegerin synthesis via potentiation of p38 MAP kinase and SAPK/JNK in osteoblasts. Our present findings could present a new significant aspect in the favorable effect of EGCG on the prevention of osteoporotic bone loss and fracture especially in elderly people since osteoprotegerin secreted from osteoblasts is well-recognized to act as a suppressor of osteoclastic bone resorption.

Incretins amplify TNF-α-stimulated IL-6 synthesis in osteoblasts: Suppression of the IκB/NF-κB pathway.

Incretins including glucagon-like peptide-1 (GLP-1) and glucose­dependent insulinotropic polypeptide (GIP) secreted from the small intestine after oral food ingestion are currently recognized to stimulate insulin secretion from pancreatic ß cells. We previously reported that p70 S6 kinase limits the tumor necrosis factor­α (TNF­α)­stimulated interleukin-6 (IL­6) synthesis in osteoblast­like MC3T3­E1 cells. In the present study, we investigated the effects of incretins on the TNF­α­induced IL­6 synthesis and the underlying mechanism in MC3T3­E1 cells. GLP­1 and GIP significantly upregulated both TNF­α­stimulated IL­6 release and mRNA levels. Wedelolactone, an inhibitor of IκB kinase, amplified the TNF-α-induced IL­6 release. GLP­1 significantly attenuated the TNF­α­induced phosphorylation of IκB without affecting the phosphorylation of p70 S6 kinase. On the other hand, GLP­1 markedly induced the phosphorylation of cAMP response element-binding protein (CREB). H­89, an inhibitor of protein kinase A, significantly suppressed the enhancement by GLP-1 of TNF-α-stimulated IL­6 release. Dibutyryl cAMP, a permeable analogue of cAMP, which suppressed the TNF-α-induced IκB phosphorylation, amplified the IL­6 release. These results strongly suggest that incretins upregulate the TNF-α-stimulated IL­6 synthesis in osteoblasts, and that the amplifying effect of incretin is exerted via reducing the IκB/NF­κB pathway through the adenylyl cyclase-cAMP system.

AICAR reduces the collagen-stimulated secretion of PDGF-AB and release of soluble CD40 ligand from human platelets: Suppression of HSP27 phosphorylation via p44/p42 MAP kinase.

We have previously reported that collagen-induced phosphorylation of heat shock protein (HSP) 27 via p44/p42 mitogen-activated protein (MAP) kinase in human platelets is sufficient to induce the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble cluster of differentiation 40 ligand (sCD40L). Adenosine monophosphate-activated protein kinase (AMPK), which is known to regulate energy homeostasis, has a crucial role as an energy sensor in various eukaryotic cells. The present study investigated the effects of 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranosyl 5'-monophosphate (AICAR), which is an activator of AMPK, on the collagen-induced activation of human platelets. It was demonstrated that AICAR dose-dependently reduced collagen-stimulated platelet aggregation up to 1.0 µM. Analysis of the size of platelet aggregates demonstrated that AICAR decreased the ratio of large aggregates (50-70 µm), whereas the ratio of small aggregates (9-25 µm) was increased by AICAR administration. AICAR markedly attenuated the phosphorylation levels of p44/p42 MAP kinase and HSP27, which are induced by collagen. Furthermore, AICAR significantly decreased the secretion of PDGF-AB and the collagen-induced release of sCD40L. These results indicated that AICAR-activated AMPK may reduce the secretion of PDGF-AB and the collagen-induced release of sCD40L by inhibiting HSP27 phosphorylation via p44/p42 MAP kinase in human platelets.