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// Chun-Ting Liu 1, * , Bei-Yu Wu 1, * , Yu-Chiang Hung 1, 2, * , Lin-Yi Wang 3 , Yan-Yuh Lee 3 , Tsu-Kung Lin 4 , Pao-Yen Lin 5 , Wu-Fu Chen 6 , Jen-Huai Chiang 7, 8 , Sheng-Feng Hsu 9, 10 and Wen-Long Hu 1, 11, 12, * 1 Department of Chinese Medicine, Kaohsiung Chang Gung Memorial Hospital and School of Traditional Chinese Medicine, Chang Gung University College of Medicine, Kaohsiung, Taiwan 2 School of Chinese Medicine for Post Baccalaureate, I-Shou University, Kaohsiung, Taiwan 3 Department of Physical Medicine and Rehabilitation, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 4 Department of Neurology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 5 Department of Psychiatry, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan 6 Department of Neurosurgery, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan 7 Management Office for Health Data, China Medical University Hospital, Taichung, Taiwan 8 College of Medicine, China Medical University, Taichung, Taiwan 9 Graduate Institute of Acupuncture Science, China Medical University, Taichung, Taiwan 10 Department of Chinese Medicine, China Medical University Hospital, Taipei Branch, Taipei, Taiwan 11 Kaohsiung Medical University College of Medicine, Kaohsiung, Taiwan 12 Fooyin University College of Nursing, Kaohsiung, Taiwan * These authors contributed equally to this work Correspondence to: Wen-Long Hu, email: oolonghu@gmail.com Keywords: dementia, migraine, pharmaco-epidemiology, national health insurance research database, Chinese herbal product Received: February 27, 2017 Accepted: June 28, 2017 Published: July 08, 2017 ABSTRACT Patients with migraine are reportedly at increased risk of developing dementia. We aimed to investigate the association between traditional Chinese medicine (TCM) use and dementia risk in migraine patients. This longitudinal cohort study used the Taiwanese National Health Insurance Research Database to identify 32,386 diagnosed migraine patients aged 20 years and above who received treatment from 1997 to 2010. To balance comparability between TCM users and non-TCM users, we randomly selected equal numbers from each group, and compared subgroups compiled based on combinations of age, sex, index year, and year of migraine diagnosis. All enrollees received follow-up until the end of 2013 to measure dementia incidence. We identified 1,402 TCM users and non-TCM users after frequency matching. A total of 134 subjects were newly diagnosed with dementia during the follow-up period. TCM users were significantly less likely to develop dementia than non-TCM users. The most frequently prescribed formulae and single Chinese herbal products were Jia-Wei-Xiao-Yao-San and Yan-Hu-Suo, respectively. This population-based study revealed a decreased dementia risk in migraine patients with TCM use. These findings may provide a reference for dementia prevention strategies, and help integrate TCM into clinical intervention programs that provide a favorable prognosis for migraine patients.

Pain is a major primary health care problem. Emerging studies show that inhibition of spinal microglial activation reduces pain. However, the precise mechanisms by which microglial activation contributes to nociceptive synaptic transmission remain unclear. In this study, we measured spontaneous synaptic activity of miniature excitatory postsynaptic currents (mEPSCs) in rat spinal cord superficial dorsal horn (SDH, laminae I and II) neurons. Lipopolysaccharide (LPS) and adenosine triphosphate (ATP) increased the frequency, but not amplitude, of mEPSCs in SDH neurons. Microglial inhibitors minocycline and paeonol, as well as an astrocyte inhibitor, a P2Y1 receptor (P2Y1R) antagonist, and a metabotropic glutamate receptor 5 (mGluR5) antagonist, all prevented LPS-induced enhancement of mEPSC frequency. In mouse behavioral testing, minocycline and paeonol effectively reduced acetic acid-induced writhing and LPS-induced hyperalgesia. These results indicate that LPS-activated microglia release ATP, which stimulates astrocyte P2Y1Rs to release glutamate, triggering presynaptic mGluR5 receptors and increasing presynaptic glutamate release, leading to an increase in mEPSC frequency and enhancement of nociceptive transmission in SDH neurons. We propose that these effects can serve as a new electrophysiological model for evaluating pain. Moreover, we predict that pharmacologic agents capable of inhibiting the LPS-induced enhancement of mEPSC frequency in SDH neurons will have analgesic effects.

Whole-cell patch-clamp recordings investigated the electrophysiological effects of 2'-hydroxy-4'-methoxyacetophenone (paeonol), one of the major components of Moutan Cortex, in hippocampal CA1 neurons and nucleus ambiguus (NA) neurons from neonatal rats as well as in lung epithelial H1355 cells expressing Kv2.1 or Kv1.2. Extracellular application of paeonol at 100μM did not significantly affect the spontaneous action potential frequency, whereas paeonol at 300μM increased the frequency of spontaneous action potentials in hippocampal CA1 neurons. Paeonol (300μM) significantly decreased the tetraethylammonium-sensitive outward current in hippocampal CA1 neurons, but had no effect upon the fast-inactivating potassium current (IA). Extracellular application of paeonol at 300μM did not affect action potentials or the delayed outward currents in NA neurons. Paeonol (100μM) reduced the Kv2.1 current in H1355 cells, but not the Kv1.2 current. The inhibitor of Kv2, guangxitoxin-1E, reduced the delayed outward potassium currents in hippocampal neurons, but had only minimal effects in NA neurons. We demonstrated that paeonol decreased the delayed outward current and increased excitability in hippocampal CA1 neurons, whereas these effects were not observed in NA neurons. These effects may be associated with the inhibitory effects on Kv2.1 currents.

Paeonol is a major constituent of the Chinese herb Moutan cortex radices. Recent studies report that paeonol has neuroprotective effects and improves impaired learning and memory. However, its underlying mechanisms by which paeonol contributes to synaptic transmission remain unclear. In this study, we found that paeonol increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs), but had no effect on the amplitude in rat hippocampal CA1 neurons. Similarly, the acetylcholinesterase (AChE) inhibitor rivastigmine increased the frequency of mEPSCs, but had no effect upon amplitude in rat hippocampal neurons. Rivastigmine also inhibited the delayed outward K+ currents in rat hippocampal CA1 neurons, but had no effect in nucleus ambiguus (NA) neurons. The Kv2 blocker guangxitoxin-1E increased the frequency of both mEPSCs and sEPSCs of rat hippocampal CA1 neurons, without affecting their amplitude. Our results suggest that paeonol and rivastigmine enhance spontaneous presynaptic transmitter release, which may be associated with the inhibition of the hippocampal Kv2 current and with therapeutic potential in neurotransmitter deficits found in Alzheimer's disease (AD). Moreover, our data also show that paeonol protects against Aβ25-35-induced impairment of long-term potentiation (LTP) in mouse hippocampal neurons. However, guangxitoxin-1E failed to potentiate the evoked field excitatory postsynaptic potentials (fEPSPs), LTP and Aβ25-35-induced impairment of LTP. These results indicate that paeonol may has the potential to improve learning and memory in AD. Interestingly, this effect is not involved in the inhibition of the hippocampal Kv2 current.

Electroacupuncture (EA) is a complementary therapy to improve morphine analgesia for postoperative pain, but underlying mechanism is not well-known. Herein, we investigated EA-induced analgesic effect in a plantar incision (PI) model in male Sprague-Dawley rats. PI was performed at the left hind paw. EA of 4 Hz and high intensity or sham needling was conducted at right ST36 prior to PI and repeated for another 2 days. Behavioral responses to mechanical and thermal stimuli, spinal phospho-ERK, and Fos expression were all analyzed. In additional groups, naloxone and morphine were administered to elucidate involvement of opioid receptors and for comparison with EA. EA pretreatment significantly reduced post-PI tactile allodynia for over 1 day; repeated treatments maintained analgesic effect. Intraperitoneal naloxone could reverse EA analgesia. Low-dose subcutaneous morphine (1 mg/kg) had stronger inhibitory effect on PI-induced allodynia than EA for 1 h. However, analgesic tolerance appeared after repeated morphine injections. Both EA and morphine could equally inhibit PI-induced p-ERK and Fos inductions. We conclude that though EA and morphine attenuate postincision pain through opioid receptor activations, daily EA treatments result in analgesic accumulation whereas daily morphine injections develop analgesic tolerance. Discrepant pathways and mechanisms underlying two analgesic means may account for the results.

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Integrins are the major adhesive molecules in mammalian cells and have been associated with metastasis of cancer cells. Insulin-like growth factor-I (IGF)-I plays an important role in regulating cell growth, proliferation, survival, and metabolism. However, the effects of IGF-I in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that IGF-I increased the migration and the expression of α5β1 integrin in human chondrosarcoma cells. Pretreatment of cells with IGF-I receptor antibody reduced IGF-I-induced cell migration and integrin expression. Activations of phosphatidylinositol 3-kinase (PI3K), Akt, and nuclear factor-κB (NF-κB) pathways after IGF-I treatment were demonstrated, and IGF-I-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of PI3K, Akt, and NF-κB cascades. Taken together, our results indicated that IGF-I enhances the migration of chondrosarcoma cells by increasing α5β1 integrin expression through the IGF-I receptor/PI3K/Akt/NF-κB signal transduction pathway. J. Cell. Physiol. 226: 3270–3277, 2011. © 2011 Wiley Periodicals, Inc.

The use of anesthetics to stabilize animals for the purpose of electroacupuncture (EA) analgesic studies can be problematic because of the interference of differential physiological responses to EA and pain. In this study, EA-induced physiological profiles were surveyed under a sub-minimal alveolar concentration (sub-MAC) of two different anesthetics in a previously proposed minimal stress model. First, to select an adequate concentration, compliance with EA and tail-flick stimulation was evaluated under various concentrations of halothane and isoflurane. Second, using the chosen concentrations, low- (4-Hz) and high-frequency (100-Hz) EA were conducted on the right hind limb. The EA effects of the two gases were compared by tail-flick latency (TFL), hemodynamic variables, and individual variations in analgesic sensitivity. The optimal concentrations for halothane and isoflurane were 0.5% and 0.75%, respectively. TFLs were stable under these anesthetic levels, but rats under 0.75% isoflurane had better compliance than those under 0.5% halothane. EA inhibited TFLs with distinct analgesic patterns when comparing high- and low-frequency EA, but TFL suppression did not differ between the two gases. Heart rate and blood pressure showed temporal and differential responses to low- vs. high-frequency EA, but were comparable between groups under the two anesthetics. The ratios of EA non-responders in the isoflurane and halothane groups were 32.4% and 26.7%, respectively, without statistical difference. We concluded that sub-MAC halothane and isoflurane provide optimal conditions for the study of EA-induced analgesia in rats. In this model, 0.75% isoflurane appears to be a better choice than 0.5% halothane in terms of EA compliance.

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. This study is the first to investigate the anti-cancer effects of the new benzimidazole derivative (5-methyl-2(pyridine-3-yl)-1-(3,4,5-trimethoxybenzyl)benzimidazole; MPTB) in human chondrosarcoma cells. MPTB-induced cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353 but not in primary chondrocytes. MPTB-induced upregulation of Bax and Bak and dysfunction of mitochondria in chondrosarcoma. MPTB triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol calcium levels, and increased glucose-regulated protein (GRP) expression. MPTB also increased calpain expression. Transfection of cells with GRP78 or calpain siRNA reduced MPTB-mediated cell apoptosis in JJ012 cells. Importantly, animal studies have revealed a dramatic 44% reduction in tumor volume after 21 d of treatment. This study demonstrates novel anti-cancer activity of MPTB against human chondrosarcoma cells and in murine tumor models. Mol. Carcinog. ©2011 Wiley-Liss, Inc.

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which is abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and cell proliferation via interaction with its receptor, alphavbeta3 integrin. However, the effect of OPN on migration activity in human lung cancer cells is mostly unknown. Here we found that OPN increased the migration via activation of alphavbeta3 integrin in human lung cancer cells (A549 cells). Phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002), Akt inhibitor or ERK inhibitor (PD98059) inhibited the OPN-induced increase in the migration of lung cancer cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), p85 subunit of PI3K, serine 473 of Akt and ERK. In addition, treatment of A549 cells with NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) inhibited OPN-induced migration of lung cancer cells. Stimulation of A549 cells with OPN also induced IkappaB kinase alpha/beta (IKK alpha/beta) phosphorylation, IkappaBalpha phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. The OPN-mediated increases in IKK alpha/beta, IkappaBalpha and p65 Ser(536) phosphorylation were inhibited by Ly294002, Akt inhibitor and PD98059. Co-transfection with FAK, p85, Akt and ERK mutants also reduced the OPN-induced kappaB-luciferase activity. Taken together, these results suggest that OPN acts through alphavbeta3 integrin, which in turn activates the FAK, PI3K, Akt, ERK and NF-kappaB pathways, contributing to the migration of lung cancer cells.

Transforming growth factor-beta1 (TGF-beta1) plays a crucial role in adhesion and migration of human cancer cells. Besides, integrins are the major adhesive molecules in mammalian cells. Here we found that TGF-beta1 increased the migration and cell surface expression of beta1 integrin in human lung cancer cells (A549 cells). TGF-beta1 stimulation increased phosphorylation of p85alpha subunit of phosphatidylinositol 3-kinase (PI3K) and Ser(473) of Akt was determined. Besides, we performed that PI3K inhibitor (Ly294002) or Akt inhibitor suppressed the TGF-beta1-induced migration activities of A549 cells. Treatment of A549 cells with NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also repressed TGF-beta1-induced cells migration and beta1 integrins expression. In addition, treatment of A549 cells with TGF-beta1 induced IkappaB kinase alpha/beta (IKKalpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the TGF-beta1-mediated increases in IKKalpha/beta, IkappaBalpha phosphorylation and p65 Ser(536) phosphorylation were inhibited by Ly294002 and Akt inhibitor. Co-transfection with p85alpha and Akt mutants also reduced the TGF-beta1-induced kappaB-luciferase activity. Taken together, our results suggest that TGF-beta1 acts through PI3K/Akt, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of beta1 integrins and contributing the migration of human lung cancer cells.

Background. Electrical stimulation of damaged peripheral nerve may aid regeneration. Objective. The purpose of this study was to determine whether 1 mA of percutaneous electrical stimulation at 1, 2, 20, or 200 Hz augments regeneration between the proximal and distal nerve stumps. Methods. A10-mm gap was made in rat sciatic nerve by suturing the stumps into silicone rubber tubes. A control group received no stimulation. Starting 1 week after transection, electrical stimulation was applied between the cathode placed at the distal stump and the anode at the proximal stump every other day for 6 weeks. Results. Higher frequency stimulation led to less regeneration compared to lower frequencies. Quantitative histology of the successfully regenerated nerves revealed that the groups receiving electrical treatment, especially at 2 Hz, had a more mature structure with a smaller cross-sectional area, more myelinated fibers, higher axon density, and higher ratio of blood vessel to total nerve area compared with the controls. Electrophysiology showed significantly shorter latency, longer duration, and faster conduction velocity. Conclusion. Electrical stimulation can have either a positive or negative impact on peripheral nerve regeneration. Clinical trials that combine stimulation with rehabilitation must determine the parameters that are most likely to be safe and effective.

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. However, the precise molecular events generated by US in chondrocytes have not been clarified well. Here we found that US stimulation transiently increased the surface expression of alpha2, alpha5, beta1 or beta3 but not alpha3 or alpha4 integrins in human chondrocytes, as shown by flow cytometric analysis. US stimulation increased prostaglandin E(2) formation as well as the protein and mRNA levels of cyclooxygenase-2 (COX-2). At the mechanistic level, anti-integrin beta1 and beta3 antibodies or beta1 and beta3 integrin small interference RNA attenuated the US-induced COX-2 expression. Integrin-linked kinase (ILK) inhibitor (KP-392), Akt inhibitor, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited the potentiating action of US. US stimulation promotes kinase activity of ILK, phosphorylation of Akt. In addition, US stimulation also induces IKKalpha/beta phosphorylation, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. The binding of p65 to the NF-kappaB element, as well as the recruitment of p300 and the enhancement of p50 acetylation on the COX-2 promoter was enhanced by US. Taken together, our results provide evidence that US stimulation increases COX-2 expression in chondrocytes via the integrin/ILK/Akt/NF-kappaB and p300 signaling pathway.

Urinary stone disease is a common disease and has a high rate of recurrence. There is no ideal long-term medical treatment to prevent the recurrence of urinary stones. Wu-Ling-San (WLS) formula has been used for centuries in China for long-term treatment of urological diseases. However, no pharmacological studies have been conducted to evaluate its effect on urinary stone disease. Therefore, using a photospectrometer, we studied the effects of WLS on nucleation, growth and aggregation of calcium oxalate in vitro. The results showed that WLS extract significantly slowed the speed of calcium oxalate ( CaOx ) crystal nucleation. WLS extracts at concentrations of 6.25, 12.5, 25, and 50 mg/ml inhibited nucleation of calcium oxalate crystallization by 344, 387, 543, and 943%, respectively. WLS extracts did not inhibit the growth of CaOx crystallization; however, WLS extracts at concentrations of 12.5 and 25 mg/ml significantly inhibited the aggregation of CaOx crystallization by 74.24% and 75.05%, respectively. WLS extract at a concentration of 50 mg/ml inhibited CaOx aggregation by 92.49%. In conclusion, our results indicate that WLS extract inhibited calcium oxalate nucleation and aggregation, and may have the potential to prevent stone recurrence.

Traditional Chinese herbal products (CHPs) have been described in ancient medicine systems as treatments for various stroke-associated ailments. This study is aimed to investigate the prescription patterns and combinations of CHPs for ischemic stroke in Taiwan. Prescriptions of CHPs for ischemic stroke were obtained from the National Health Insurance Research Database (NHIRD) of Taiwan. Every prescription with a leading diagnosis of ischemic stroke made during 2000–2010 was analyzed. Descriptive statistics were applied to the pattern of co-prescriptions. Multiple logistic regression models were used to assess demographic and risk factors that are correlated with CHP use. The dataset of inpatient claims data contained information on 15,896 subjects who experienced ischemic stroke from 2000 to 2010. There was an average of 5.82 CHPs in a single prescription for subjects with ischemic stroke. Bu-yang-huan-wu-tang (BYHWT) (40.32%) was by far the most frequently prescribed formula CHP for ischemic stroke, and the most commonly used combination of two-formula-CHP was BYHWT with Shu-jin-huo-xue-tang (SJHXT) (4.40%). Dan Shen (16.50%) was the most commonly used single CHP for ischemic stroke, and the most commonly used combination of two single CHPs was Shi Chang Pua with Yuan Zhi (4.79%). We found that BYHWT and Dan Shen were the most frequently prescribed formula and single CHP for ischemic stroke, respectively. These results provide information about individualized therapy and may contribute to further pharmacologic experiments and clinical trials.

To investigate the efficacy of stimulation at the Jing-Well points of meridians in advanced clinical practice.

Our previous studies showed that Gastrodia elata (GE), an herb used in traditional Chinese medicine, has both anti-convulsive and free radical-scavenging activities in kainic acid (KA)-treated rats. The aim of the present study was to further investigate possible physiological mechanisms of GE against activities of neuronal nitric oxide synthase (nNOS) and microglia in KA-treated rats; 0.5 g/kg and 1.0 g/kg of GE extract were administered orally, whereas 20 mg/kg of N-nitro-L-arginine methyl ester (L-NAME) was administered intraperitoneally (ip), both at 30 minutes prior to KA (2 μg/2 μl) being injected into the right hippocampus region of rats. ED1-staining, apoptotic, inducible nitric oxide synthase (iNOS), and nNOS-staining cells were observed in the hippocampus region. The results indicated that 1.0 g/kg of GE and 20 mg/kg of L-NAME reduced the counts of ED1-stained cells, and 0.5 g/kg and 1.0 g/kg of GE, and 20 mg/kg of L-NAME reduced the numbers of apoptotic cells and nNOS-staining cells. In addition, 20 mg/kg of L-NAME also reduced the numbers of iNOS-staining cells, but 0.5 g/kg and 1.0 g/kg of GE did not. This study demonstrated that GE was able to reduce nNOS, microglia activation and apoptosis, suggesting that GE has a protective effect against neuronal damage in KA-treated rats.

The aim of this study was to investigate the effect of electrical stimulation on activator protein-1 (AP-1) in recombinant liver cells. In order to elucidate the molecular effects of electrical stimulation on cells, AP-1 expression was detected by a luciferase assay. The parameters used were taken from clinical electroacupuncture (EA) therapy as follows: biphasic rectangular symmetrical pulses (frequency: 2, 10 and 100 Hz; pulse width: 50, 80, 130 and 250 μsec; intensity: 1, 2, 5 and 10 mA; time: 5, 10, 20 and 30 minutes). S (10% fetal bovine serum in medium), SF (serum free medium) and 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment represented the experimental, negative and positive groups, respectively. We found that electrical stimulation with 10 Hz, a pulse width of 130 μsec, and a duration of 30 minutes gave a significant increase in AP-1 activity. In contrast, the intensity of the stimulation had no significant effect on AP-1 activity. In conclusion, our data suggest that electrical stimulation with an appropriate frequency, duration and pulse width could cause an increase in AP-1 activity in cells.

The purpose of this study was to evaluate the effectiveness of acupressure on gastrointestinal (GI) motility in women after trans-abdominal hysterectomy (TAH). Patients were randomly assigned into two groups of 21 and 20 patients each. The experimental group received acupressure for 3 minutes at each of three meridian points: Neiguan (PC-6), Zusanli (ST-36) and Sanyinjiao (SP-6). The control group received 3 minutes of acupressure on sham points. Acupressure was performed twice a day. A questionnaire was used to determine patients' satisfaction prior to and after afternoon acupressure. GI contractions were measured with a multifunctional stethoscope before and after acupressure. Acupressure of these three meridian points significantly (p < 0.05) increased GI motility in the experimental group, but there was little change in the control group (p > 0.05). Our conclusions are that non-invasive acupressure of these meridian points can significantly improve GI motility and can be incorporated into the technical curriculum and clinical education program of nursing schools. Patients and their family members can be taught to continue this procedure at home to enhance GI motility in patients who have undergone TAH.

NCTD is a demethylated form of cantharidin with antitumor properties, which is now in use as a routine anticancer drug against hepatoma. However, there is limited information on the effect of NCTD on human cancer cells. In the present study, NCTD inhibited proliferation, caused mitotic arrest, then progressed to apoptosis within 96 hr in 3 human hepatoma cell lines: HepG2, Hep3B and Huh-7. NCTD treatment (5 microg/ml) enhanced the expression of Cdc25C and p21(Cip1/Waf1), increasing the phosphorylation of these 2 proteins. In addition, NCTD treatment induced an earlier increase in cyclin B1-associated histone H1 kinase activity within 48 hr, but an approximately 70% reduction of both protein level and kinase activity of cyclin B1 was observed at 72 hr. Treatment with NCTD significantly decreased the expression of p53 protein but did not affect the expression of Cdk1 and p27(Kip1). Moreover, NCTD treatment also increased the phosphorylation of Bcl-2 and Bcl-X(L) but did not affect the expression of Bax or Bad. Bcl-2 phosphorylation appears to inhibit its binding to Bax since less Bax was detected in immunocomplex with Bcl-2 in NCTD-treated HepG2 cells. In addition, NCTD treatment caused activation of caspase-9 and caspase-3, preceding DNA fragmentation and morphologic features of apoptosis. Pretreatment with the broad-spectrum caspase inhibitor z-VAD-fmk markedly inhibited NCTD-induced caspase-3 activity and cell death. These results suggest that phosphorylation of p21(Cip1/Waf1) and Cdc25C and biphasic regulation of cyclin B1-associated kinase activity may contribute to NCTD-induced M-phase cell-cycle arrest. Furthermore, the increase of p21(Cip1/Waf1), phosphorylation of Bcl-2 and Bcl-X(L), activation of caspase-9 and caspase-3 may be the molecular mechanism through which NCTD induces apoptosis.

Aims Postoperative pain is a major problem. Electroacupuncture (EA) has been accepted as a useful and low-risk complementary therapy for post-operative pain. Animal studies indicate that surgical incision activates p38 MAPK in the spinal microglia, which critically contributes to post-incisional nociceptive development. How EA affects incision-induced p38 activation is important but yet to be fully elucidated. Methods Male adult rats received plantar incision (PI) at the right hind paw followed by 30-min EA of 4-Hz, one of two intensities (3 and 10 mA), and at right ST36 (Zusanli) acupoint immediately after PI and for 3 successive days. EA analgesia was evaluated by von Frey fibers and Hargreaves' tests. Spinal p38 activation was examined by immunostaining. In separate groups, SB203580, a p38 inhibitor, was intrathecally injected alone or with EA to test the combining effect on nociception and spinal phospho-p38. Key findings EA of 10-mA significantly ameliorated mechanical allodynia, but 3-mA did not. None of them altered thermal hyperalgesia. Repeated EA could not inhibit phospho-p38 in the PI rats, contrarily, EA per se significantly induced phospho-p38 in the normal rats. Intrathecal SB203580 injection dose-dependently prevented PI-induced allodynia. Combination of low-dose SB203580 and 3-mA EA, which were ineffective individually, profoundly reduce post-PI allodynia. Significance We demonstrated that 10-mA EA exerts a significant inhibition against post-PI mechanical hypersensitivity via a p38-independent pathway. Importantly, co-treatment with low-dose p38 inhibitor and 3-mA EA can counteract spinal phospho-p38 to exert strong analgesic effect. Our finding suggests a novel strategy to improve EA analgesic quality.

Objective To investigate the clinical effects of laser acupuncture therapy for temporomandibular disorders (TMD) after ineffective previous treatments. Methods A retrospective observational study was conducted in 29 treatment-resistant TMD patients (25 women, 4 men; age range, 17–67 years). Subjects were treated 3 times per week for 4 weeks with the Handylaser Trion (GaAlAs laser diode, 810 nm, 150 mW, pulsed waves), which delivered 0.375 J of energy (5 s) to ST7, ST6, and LI4 and 3 J (40 s) to each Ashi point, 7.5–26.25 J/cm2 in total. The visual analog scale (VAS) and maximal mouth opening (MMO) were evaluated before and after treatment. Results VAS analysis showed that the patients were free of pain at rest (endpoint) after 5.90±6.08 sessions of laser acupuncture for acute TMD and after 16.21±17.98 sessions for chronic TMD. The VAS score on palpation of the temporomandibular joint reduced to 0.30±0.67 for patients with acute TMD (p = 0.005) and to 0.47±0.84 for those with chronic TMD (p

Background Indoxyl sulfate is considered to play a pathological role in the progression of chronic kidney disease. The aim of this study was to investigate the deleterious effects of indoxyl sulfate on kidney mesangial cells. Materials and methods Rat renal mesangial cells were exposed to indoxyl sulfate at a serial concentrations. Cytotoxicity of indoxyl sulfate on renal mesangial cells was determined using MTT assay. Protein levels of cleaved caspase-3, angiotensin, angiotensin converting enzyme (ACE) and renin were detected by immunoblotting. Reverse transcriptional PCR was performed to determine the mRNA expression. Results Level of cleaved caspase-3 was augmented while the cell viability was inhibited by indoxyl sulfate in a dose-dependent manner. The mRNA expressions of pro-renin and ACE were upregulated in mesangial cells exposed to indoxyl sulfate. Level of renin and ACE was increased in response to indoxyl sulfate exposure in time-dependent fashion. Conclusion Indoxyl sulfate increased viability and induced cell death of renal mesangial cells, which is time-dependent. The loss of cell viability is attributed to caspase-3 activity through apoptosis pathway. RAS in renal mesangial cells is activated in response to indoxyl sulfate treatment.

This study investigated the effects of acupuncture on carbon tetrachloride (CCl4) induced acute liver injury in male rats (n = 36). The experimental groups were injected with CCl4before, during, or after acupuncture therapy. Acupoints similar to the human Tsu-San-Li (St-36) and Tai-Chung (Li-3) were needled bilaterally. Rats treated with CCl4had higher levels of serum glutamate-oxalate-transaminase (sGOT) and serum glutamate-pyruvate-transaminase (sGPT). Comparing the experimental groups, biochemical and pathological parameters of liver injury were significantly reduced when rats were acupunctured after, not before, CCl4-induced hepatotoxicity. Acupuncture at the Tsu-Sa-Li and Tai-Chung acupoints cannot prevent acute liver injury but may be effective in treating liver injury induced by carbon tetrachloride in rats.

This paper presents the evident effects of acupoint stimulation, using EEG (electroencephalogram) measurements. With acupuncture stimulation and the EEG measurement on the same meridian, EEG is able to accurately detect the effects of acupunctural point stimulation on brain waves. In this study, 24 subjects without heart or neural diseases were randomly separated into two groups of 12, named test and control groups. Similar procedures are performed; the subjects lay on a bed with eyes closed for ten minutes as the baseline. The test group received acupuncture at their Waiguan points (TE5) on their left hands for 20 minutes, while the control group did not. EEGs are recorded during pre-acupuncture, acupuncture stimulation and post-acupuncture stimulation periods. The EEG electrodes are at the T3, T4, O1 and O2 locations. Continuous wavelet transformation analysis is adopted; therefore, EEGs are divided into the following bands: δ (0.5–4HZ), θ (4–8HZ), α (8–13HZ) and β (13–30HZ). During acupuncture stimulation, the θ energy is increased and had statistical differences at all electrode points, T3, T4, O1 and O2. Upon removing the needle, the energy at the T3 and T4 points slowly declined and revealed obvious statistical differences. During acupuncture, only α energy has been noted to have statistical difference and it was increased at the T3 point. However, the energy was decreased and had no statistical difference after five minutes. Acupuncture is proven to be able to affect brain waves, as the stimulation might have changed the tissues between the cranium and scalp; therefore, the brain waves are detected more easily.

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. This study is the first to investigate the anti-cancer effects of the new benzimidazole derivative (5-methyl-2(pyridine-3-yl)-1-(3,4,5-trimethoxybenzyl)benzimidazole; MPTB) in human chondrosarcoma cells. MPTB-induced cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353 but not in primary chondrocytes. MPTB-induced upregulation of Bax and Bak and dysfunction of mitochondria in chondrosarcoma. MPTB triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol calcium levels, and increased glucose-regulated protein (GRP) expression. MPTB also increased calpain expression. Transfection of cells with GRP78 or calpain siRNA reduced MPTB-mediated cell apoptosis in JJ012 cells. Importantly, animal studies have revealed a dramatic 44% reduction in tumor volume after 21 d of treatment. This study demonstrates novel anti-cancer activity of MPTB against human chondrosarcoma cells and in murine tumor models.

Bradykinin (BK) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. BK has recently been shown to be involved in carcinogenesis and cancer progression. In this study, we found that BK increased the migration and the expression of alpha2beta1 integrin in human chondrosarcoma cells. We also found that human chondrosarcoma tissues had significantly higher expression of the B1 and B2 receptors comparing to normal cartilage. BK-mediated migration and integrin up-regulation was attenuated by B1 and B2 BK receptor siRNA or antagonist. Activations of phospholipase C (PLC), protein kinase Cdelta (PKCdelta), and NF-kappaB pathways after BK treatment was demonstrated, and BK-induced integrin expression and migration activity was inhibited by the specific inhibitor of PLC, PKCdelta, and NF-kappaB cascades. Taken together, our results indicated that BK enhances the migration of chondrosarcoma cells by increasing alpha2beta1 integrin expression through the BK receptors/PLC/PKCdelta/NF-kappaB signal transduction pathway.

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. In the present study, we investigated the anti-cancer effect of a honokiol, an active component isolated and purified from the Magnolia officinalis in human chondrosarcoma cells. Honokiol-induced cell apoptosis in human chondrosarcoma cell lines (including: JJ012 and SW1353) but not primary chondrocytes. Honokiol also induces upregulation of Bax and Bak, downregulation of Bcl-XL and dysfunction of mitochondria in chondrosarcoma cells. Honokiol triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol-calcium levels. We also found that honokiol increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced honokiol-mediated cell apoptosis in JJ012 cells. Importantly, animal studies have revealed a dramatic 53% reduction in tumor volume after 21 days of treatment. This study demonstrates that honokiol may be a novel anti-cancer agent targeting chondrosarcoma cells.

Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. However, the effect of Cyr61 on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that Cyr61 increased the migration and expression of matrix metalloproteinase (MMP)-13 in human chondrosarcoma cells (JJ012 cells). RGD peptide, alphavbeta3 monoclonal antibody and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the Cyr61-induced increase of the migration and MMP-13 upregulation of chondrosarcoma cells. Cyr61 stimulation increased the phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). In addition, activator protein-1 (AP-1) decoy oligodeoxynucleotide also suppressed the MMP-13 messenger RNA and enzyme activity enhanced by Cyr61. Moreover, Cyr61 increased the binding of c-Fos and c-Jun to the AP-1 element on the MMP-13 promoter. Taken together, our results indicated that Cyr61 enhances the migration of chondrosarcoma cells by increasing MMP-13 expression through the alphavbeta3 integrin receptor, FAK, ERK, c-Fos/c-Jun and AP-1 signal transduction pathway.

Bone morphogenetic protein-2 (BMP-2), a member of transforming growth factor-beta superfamily, plays a crucial role in migration and metastasis of human cancer cells. Integrins are the major adhesive molecules in mammalian cells. Here we found that BMP-2 directed the migration and increased cell surface and mRNA expression of beta1 integrin in human chondrosarcoma cancer cells (JJ012). Pretreated of JJ012 cells with phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002) or Akt inhibitor inhibited the BMP-2-mediated migration and integrin expression. BMP-2 increased the phosphorylation of p85 subunit of PI3K and serine 473 of Akt. In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited BMP-2-mediated cells migration and integrin upregulation. Stimulation of JJ012 cells with BMP-2 induced IkappaB kinase (IKKalpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the BMP-2-mediated increasing of IKKalpha/beta phosphorylation, IkappaB phosphorylation, and p65 Ser(536) phosphorylation were inhibited by Ly294002 and Akt inhibitor. Co-transfection with p85 and Akt mutants also reduced the BMP-2-induced kappaB-luciferase activity. Taken together, these results suggest that the BMP-2 acts through PI3K/Akt, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of beta1 integrin and contributing the migration of human chondrosarcoma cells.

Aim: Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. The aim of the present study was to investigate the effect of alendronate, a bisphosphonate, on the invasion and migration of human chondrosarcoma cells (JJ012). Methods: JJ012 cells were treated with alendronate of various concentrations up to 100 μmol/L for a specified period, and then gelatin zymography and matrigel invasion assay was performed to study the effects of alendronate on matrix metalloproteinase (MMP)-2 activity and the invasion ability of JJ012 cells, respectively. Results: Our data showed that alendronate exerted a dose- and time-dependent inhibitory effect on the invasion and migration of JJ012 cells. Furthermore, gelatin zymography and RT-PCR showed that alendronate treatment decreased the activity and mRNA levels of MMP-2 in a concentration-dependent manner. Conclusion: Our findings suggest that alendronate may reduce MMP-2 secretion at the transcriptional and translational levels, and inhibit the invasion of chondrosarcoma cell. Therefore, alendronate may be a potential candidate for the systemic therapy of chondro-sarcomas, as well as other malignant diseases.

2-Methoxyestradiol (2ME) is an endogenous metabolite with estrogen receptor-independent anti-tumor activity. The current study seeks to determine the mechanism of anti-tumor activity of 2ME on human chondrosarcoma. 2ME caused a time- and dose-dependent cytotoxity in chondrosarcoma cells, while primary chondrocytes were minimally affected. Cells accumulated in G0/G1 phase in response to 2ME and DAPI stain indicated an induction of apoptosis. Bax, Cytochrome C, and Caspase-3 protein expression were increased, while p53 expression was decreased. A higher Bax/Bcl-2 ratio followed 2ME treatment. 2ME has a potentially promising role as a systemic therapy of chondrosarcoma when the mechanism of action is better delineated.

The purpose of this study was to determine whether low-power pulsed laser irradiation could affect the regeneration of a 10-mm gap of rat sciatic nerve created between the proximal and distal nerve stumps, which were sutured into silicone rubber tubes. After 8 weeks of recovery, pulsed laser-irradiated groups at frequencies of 5 kHz and 20 kHz both had significantly lower success percentages of regeneration (50% and 44%, respectively) compared to sham-irradiated controls (100%). In addition, qualitative and quantitative histology of the regenerated nerves revealed a less mature ultrastructural organization with a smaller cross-sectional area and a lower number of myelinated axons in both pulsed laser-irradiated groups than in controls. These results suggest that pulsed laser irradiation could elicit suppressing effects on regenerating nerves.