Yi Wen Lin

Dipeptidyl peptidase-4 inhibitor improves neovascularization by increasing circulating endothelial progenitor cells

BACKGROUND AND PURPOSE Current methods used to treat critical limb ischaemia (CLI) are hampered by a lack of effective strategies, therefore, therapeutic vasculogenesis may open up a new field for the treatment of CLI. In this study we investigated the ability of the DPP‐4 inhibitor, sitagliptin, originally used as a hypoglycaemic agent, to induce vasculogenesis in vivo.

GroEL1, a Heat Shock Protein 60 of Chlamydia pneumoniae, Induces Lectin-Like Oxidized Low-Density Lipoprotein Receptor 1 Expression in Endothelial Cells and Enhances Atherogenesis in Hypercholesterolemic Rabbits

Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) plays a major role in oxidized low-density lipoprotein-induced vascular inflammation. Chlamydia pneumoniae has been found in atherosclerotic lesions and is related to atherosclerotic pathogenesis, although its specific mechanism remains unknown. This study was conducted to investigate the mechanisms of LOX-1 expression in GroEL1 (a heat shock protein from C. pneumoniae)-administered human coronary artery endothelial cells (HCAECs) and atherogenesis in hypercholesterolemic rabbits. We demonstrated that in the hypercholesterolemic rabbit model, GroEL1 administration enhanced fatty streak and macrophage infiltration in atherosclerotic lesions, which may be mediated by elevated LOX-1 expression. In in vitro study using HCAECs, stimulation with GroEL1 increased TLR4 and LOX-1 expression. Increased LOX-1 expression was downregulated by Akt activation and PI3K-mediated endothelial NO synthase activation. PI3K inhibitor and NO synthase inhibitor induced LOX-1 mRNA production, whereas the NO donor ameliorated the increasing effect of LOX-1 mRNA in GroEL1-stimulated HCAECs. LOX-1 expression was regulated by NADPH oxidase, which mediates reactive oxygen species production and intracellular MAPK signaling pathway in GroEL1-stimulated HCAECs. Treatment with polyethylene-glycol–conjugated superoxide dismutase, apocynin, or diphenylene iodonium significantly decreased GroEL1-induced LOX-1 expression, as did the knockdown of Rac1 gene expression by RNA interference. In conclusion, the GroEL1 protein may induce LOX-1 expression in endothelial cells and atherogenesis in hypercholesterolemic rabbits. The elevated level of LOX-1 in vitro may be mediated by the PI3K–Akt signaling pathway, endothelial NO synthase activation, NADPH oxidase-mediated reactive oxygen species production, and MAPK activation in GroEL1-stimulated HCAECs. The GroEL1 protein of C. pneumoniae may contribute to vascular inflammation and cardiovascular disorders.

Analgesic Effect of Electroacupuncture in a Mouse Fibromyalgia Model: Roles of TRPV1, TRPV4, and pERK.

Fibromyalgia (FM) is among the most common chronic pain syndromes encountered in clinical practice, but there is limited understanding of FM pathogenesis. We examined the contribution of transient receptor potential vanilloid 1 (TRPV1) and TRPV4 channels to chronic pain in the repeated acid injection mouse model of FM and the potential therapeutic efficacy of electroacupuncture. Electroacupuncture (EA) at the bilateral Zusanli (ST36) acupoint reduced the long-lasting mechanical hyperalgesia induced by repeated acid saline (pH 4) injection in mouse hindpaw. Isolated L5 dorsal root ganglion (DRG) neurons from FM model mice (FM group) were hyperexcitable, an effect reversed by EA pretreatment (FM + EA group). The increase in mechanical hyperalgesia was also accompanied by upregulation of TRPV1 expression and phosphoactivation of extracellular signal regulated kinase (pERK) in the DRG, whereas DRG expression levels of TRPV4, p-p38, and p-JNK were unaltered. Blockade of TRPV1, which was achieved using TRPV1 knockout mice or via antagonist injection, and pERK suppressed development of FM-like pain. Both TRPV1 and TRPV4 protein expression levels were increased in the spinal cord (SC) of model mice, and EA at the ST36 acupoint decreased overexpression. This study strongly suggests that DRG TRPV1 overexpression and pERK signaling, as well as SC TRPV1 and TRPV4 overexpression, mediate hyperalgesia in a mouse FM pain model. The therapeutic efficacy of EA may result from the reversal of these changes in pain transmission pathways.

Abundant expression and functional participation of TRPV1 at Zusanli acupoint (ST36) in mice: mechanosensitive TRPV1 as an “acupuncture-responding channel”

BackgroundAcupuncture is a therapy that involves applying mechanical stimulation to acupoints using needles. Although acupuncture is believed to trigger neural regulation by opioids or adenosine, still little is known about how physical stimulation is turned into neurological signaling. The transient receptor potential vanilloid receptors 1 and 4 (TRPV1 and TRPV4) and the acid-sensing ion channel 3 (ASIC3) are regarded as mechanosensitive channels. This study aimed to clarify their role at the Zusanli acupoint (ST36) and propose possible sensing pathways linking channel activation to neurological signaling.MethodsFirst, tissues from different anatomical layers of ST36 and the sham point were sampled, and channel expressions between the two points were compared using western blotting. Second, immunofluorescence was performed at ST36 to reveal distribution pattern of the channels. Third, agonist of the channels were injected into ST36 and tested in a mouse inflammatory pain model to seek if agonist injection could replicate acupuncture-like analgesic effect. Last, the components of proposed downstream sensing pathway were tested with western blotting to determine if they were expressed in tissues with positive mechanosensitive channel expression.ResultsThe results from western blotting demonstrated an abundance of TRPV1, TRPV4, and ASIC3 in anatomical layers of ST36. Furthermore, immunofluorescence showed these channels were expressed in both neural and non-neural cells at ST36. However, only capsaicin, a TRPV1 agonist, replicated the analgesic effect of acupuncture when injected into ST36. Components of calcium wave propagation (CWP, the proposed downstream sensing pathway) were also expressed in tissues with abundant TRPV1 expression, the muscle and epimysium layers.ConclusionsThe results demonstrated mechanosensitive channel TRPV1 is highly expressed at ST36 and possibly participated in acupuncture related analgesia. Since CWP was reported by other to occur during acupuncture and its components were shown here to express in tissues with positive TRPV1 expression. These findings suggest TRPV1 might act as acupuncture-responding channel by sensing physical stimulation from acupuncture and conducting the signaling via CWP to nerve terminals. This study provided a better understanding between physical stimulation from acupuncture to neurological signaling.

Acid-sensing ion channel 3 mediates peripheral anti-hyperalgesia effects of acupuncture in mice inflammatory pain

BackgroundPeripheral tissue inflammation initiates hyperalgesia accompanied by tissue acidosis, nociceptor activation, and inflammation mediators. Recent studies have suggested a significantly increased expression of acid-sensing ion channel 3 (ASIC3) in both carrageenan- and complete Freunds adjuvant (CFA)-induced inflammation. This study tested the hypothesis that acupuncture is curative for mechanical hyperalgesia induced by peripheral inflammation.MethodsHere we used mechanical stimuli to assess behavioral responses in paw and muscle inflammation induced by carrageenan or CFA. We also used immunohistochemistry staining and western blot methodology to evaluate the expression of ASIC3 in dorsal root ganglion (DRG) neurons.ResultsIn comparison with the control, the inflammation group showed significant mechanical hyperalgesia with both intraplantar carrageenan and CFA-induced inflammation. Interestingly, both carrageenan- and CFA-induced hyperalgesia were accompanied by ASIC3 up-regulation in DRG neurons. Furthermore, electroacupuncture (EA) at the ST36 rescued mechanical hyperalgesia through down-regulation of ASIC3 overexpression in both carrageenan- and CFA-induced inflammation.ConclusionsIn addition, electrical stimulation at the ST36 acupoint can relieve mechanical hyperalgesia by attenuating ASIC3 overexpression.

ELECTROACUPUNCTURE AT BAIHUI ACUPOINT (GV20) REVERSES BEHAVIOR DEFICIT AND LONG-TERM POTENTIATION THROUGH N-METHYL-D-ASPARTATE AND TRANSIENT RECEPTOR POTENTIAL VANILLOID SUBTYPE 1 RECEPTORS IN MIDDLE CEREBRAL ARTERY OCCLUSION RATS

Vascular dementia is one of the most important causes that account for 20-40% of all dementia cases. The aim of this study was to investigate whether electroacupuncture can reduce behavior deficit and long-term potentiation (LTP) in vascular dementia. Here we used a middle cerebral artery occlusion (MCAo) technique to induce a vascular dementia model with additional electroacupuncture (EA) manipulation. Behaviors were impaired in animals with MCAo, and similar results were observed with long-term potentiation induction. MCAo decreased the expression of LTP from 180.4±14.9% to 112.5±18.3%, suggesting that cerebral ischemia could impair the hippocampal LTP. In addition, immunostaining results showed that the expressions of N-methyl-D-aspartate receptor subtype 1 (NR1) and transient receptor potential vanilloid subtype 1 (TRPV1) receptors were significantly increased in the hippocampal CA1 areas. Noticeably, these phenomena can be reversed by 2 Hz EA at Baihui acupoint (GV20) for six consecutive days. Our results support a rescue role of 2 Hz EA for MCAo-induced behavior and LTP impairment. These results also suggest that NMDAR1 and TRPV1 may be involved in this pathway.

MK-0626, A Dipeptidyl Peptidase-4 Inhibitor, Improves Neovascularization by Increasing Both the Number of Circulating Endothelial Progenitor Cells and Endothelial Nitric Oxide Synthetase Expression

Current treatment modalities for critical limb ischemia (CLI) are of limited benefit; therefore, advances in therapeutic vasculogenesis may open an important new avenue for the treatment of CLI. This study examines the therapeutic potential of the DPP-4 inhibitor MK-0626 as a regulator of vasculogenesis in vivo. MK-0626 was administered daily to C57CL/B6 mice and eGFP-labeled bone marrow-transplanted ICR mice that had undergone hind limb ischemia surgery. Laser Doppler imaging and flow cytometry were used to evaluate the degree of neo-vasculogenesis and the number of circulating endothelial progenitor cells (EPCs), respectively. Cell surface markers of EPCs and the level of endothelial nitric oxide synthase (eNOS) were studied in the vessels. Mice that received MK-0626 had an elevated level of glucagon- like peptide-1 (GLP-1) and a decreased level of dipeptidyl peptidase-4 (DPP-4) in their plasma, in addition to an ischemia-induced increase in the level of stromal cell-derived factor-1 (SDF-1). In C57CL/B6 mice, blood flow in the ischemic limb was significantly improved by treatment with MK-0626. The number of circulating EPCs and both the synthesis and phosphorylation of eNOS were also increased in ischemic thigh muscle after MK-0626 treatment. In contrast, similar effects of MK-0626 were not observed in B6.129P2-Nos3(tm1Unc)/J mice (an eNOS knockout mouse). Additionally, MK-0626 treatment promoted the mobilization and homing of EPCs to ischemic tissue in eGFP transgenic mouse bone marrow-transplanted ICR mice. We conclude that both the number of circulating EPCs and neo-vasculogenesis are increased in response to DPP-4 inhibitor treatment and that this occurs via an eNOS-dependent mechanism. The results highlight the therapeutic vasculogenesis potential of the DPP-4 inhibitor MK-0626 using a hind limb ischemia mouse model.

Probing the Effects and Mechanisms of Electroacupuncture at Ipsilateral or Contralateral ST36–ST37 Acupoints on CFA-induced Inflammatory Pain

Transient receptor potential vanilloid 1 (TRPV1) and associated signaling pathways have been reported to be increased in inflammatory pain signaling. There are accumulating evidences surrounding the therapeutic effect of electroacupuncture (EA). EA can reliably attenuate the increase of TRPV1 in mouse inflammatory pain models with unclear signaling mechanisms. Moreover, the difference in the clinical therapeutic effects between using the contralateral and ipsilateral acupoints has been rarely studied. We found that inflammatory pain, which was induced by injecting the complete Freund’s adjuvant (CFA), (2.14 ± 0.1, p < 0.05, n = 8) can be alleviated after EA treatment at either ipsilateral (3.91 ± 0.21, p < 0.05, n = 8) or contralateral acupoints (3.79 ± 0.25, p < 0.05, n = 8). EA may also reduce nociceptive Nav sodium currents in dorsal root ganglion (DRG) neurons. The expression of TRPV1 and associated signaling pathways notably increased after the CFA injection; this expression can be further attenuated significantly in EA treatment. TRPV1 and associated signaling pathways can be prevented in TRPV1 knockout mice, suggesting that TRPV1 knockout mice are resistant to inflammatory pain. Through this study, we have increased the understanding of the mechanism that both ipsilateral and contralateral EA might alter TRPV1 and associated signaling pathways to reduce inflammatory pain.

Attenuation of TRPV1 and TRPV4 Expression and Function in Mouse Inflammatory Pain Models Using Electroacupuncture

Although pain is a major human affliction, our understanding of pain mechanisms is limited. TRPV1 (transient receptor potential vanilloid subtype 1) and TRPV4 are two crucial receptors involved in inflammatory pain, but their roles in EA- (electroacupuncture-) mediated analgesia are unknown. We injected mice with carrageenan (carra) or a complete Freunds adjuvant (CFA) to model inflammatory pain and investigated the analgesic effect of EA using animal behavior tests, immunostaining, Western blotting, and a whole-cell recording technique. The inflammatory pain model mice developed both mechanical and thermal hyperalgesia. Notably, EA at the ST36 acupoint reversed these phenomena, indicating its curative effect in inflammatory pain. The protein levels of TRPV1 and TRPV4 in DRG (dorsal root ganglion) neurons were both increased at day 4 after the initiation of inflammatory pain and were attenuated by EA, as demonstrated by immunostaining and Western blot analysis. We verified DRG electrophysiological properties to confirm that EA ameliorated peripheral nerve hyperexcitation. Our results indicated that the AP (action potential) threshold, rise time, and fall time, and the percentage and amplitude of TRPV1 and TRPV4 were altered by EA, indicating that EA has an antinociceptive role in inflammatory pain. Our results demonstrate a novel role for EA in regulating TRPV1 and TRPV4 protein expression and nerve excitation in mouse inflammatory pain models.

Oral Uncaria rhynchophylla (UR) reduces kainic acid-induced epileptic seizures and neuronal death accompanied by attenuating glial cell proliferation and S100B proteins in rats

AIM OF THE STUDY Epilepsy is a common clinical syndrome with recurrent neuronal discharges in cerebral cortex and hippocampus. Here we aim to determine the protective role of Uncaria rhynchophylla (UR), an herbal drug belong to Traditional Chinese Medicine (TCM), on epileptic rats. MATERIALS AND METHODS To address this issue, we tested the effect of UR on kainic acid (KA)-induced epileptic seizures and further investigate the underlying mechanisms. RESULTS Oral UR successfully decreased neuronal death and discharges in hippocampal CA1 pyramidal neurons. The population spikes (PSs) were decreased from 4.1 ± 0.4 mV to 2.1 ± 0.3 mV in KA-induced epileptic seizures and UR-treated groups, respectively. Oral UR protected animals from neuronal death induced by KA treatment (from 34 ± 4.6 to 191.7 ± 48.6 neurons/field) through attenuating glial cell proliferation and S100B protein expression but not GABAA and TRPV1 receptors. CONCLUSIONS The above results provide detail mechanisms underlying the neuroprotective action of UR on KA-induced epileptic seizure in hippocampal CA1 neurons.