Ho Jae Han

Bee venom injection into an acupuncture point reduces arthritis associated edema and nociceptive responses

&NA; Bee venom (BV) has traditionally been used in Oriental medicine to relieve pain and to treat inflammatory diseases such as rheumatoid arthritis (RA). While several investigators have evaluated the anti‐inflammatory effect of BV treatment, the anti‐nociceptive effect of BV treatment on inflammatory pain has not been examined. Previous studies in experimental animals suggest that the therapeutic effect of BV on arthritis is dependent on the site of administration. Because of this potential site specificity, the present study was designed to evaluate the anti‐nociceptive effect of BV injections into a specific acupoint (Zusanli) compared to a non‐acupoint in an animal model of chronic arthritis. Subcutaneous BV treatment (1 mg/kg per day) was found to dramatically inhibit paw edema caused by Freunds adjuvant injection. Furthermore, BV therapy significantly reduced arthritis‐induced nociceptive behaviors (i.e. the nociceptive scores for mechanical hyperalgesia and thermal hyperalgesia). These anti‐nociceptive/anti‐inflammatory effects of BV were observed from 12 days through 21 days post‐BV treatment. In addition, BV treatment significantly suppressed adjuvant‐induced Fos expression in the lumbar spinal cord at 3 weeks post‐adjuvant injection. Finally, injection of BV into the Zusanli acupoint resulted in a significantly greater analgesic effect on arthritic pain as compared to BV injection in to a more distant non‐acupoint. The present study demonstrates that BV injection into the Zusanli acupoint has both anti‐inflammatory and anti‐nociceptive effects on Freunds adjuvant‐induced arthritis in rats. These findings raise the possibility that BV acupuncture may be a promising alternative medicine therapy for the long‐term treatment of rheumatoid arthritis.

The water-soluble fraction of bee venom produces antinociceptive and anti-inflammatory effects on rheumatoid arthritis in rats.

We recently demonstrated that bee venom (BV) injection into the Zusanli acupoint produced a significantly more potent anti-inflammatory and antinociceptive effect than injection into a non-acupoint in a Freunds adjuvant induced rheumatoid arthritis (RA) model. However, the precise BV constituents responsible for these antinociceptive and/or anti-inflammatory effects are not fully understood. In order to investigate the possible role of the soluble fraction of BV in producing the anti-arthritic actions of BV acupuncture, whole BV was extracted into two fractions according to solubility (a water soluble fraction, BVA and an ethylacetate soluble fraction, BVE) and the BVA fraction was further tested. Subcutaneous BVA injection (0.9 mg/kg/day) into the Zusanli acupoint was found to dramatically inhibit paw edema and radiological change (i.e. new bone proliferation and soft tissue swelling) caused by Freunds adjuvant injection. BVA treatment also reduced the increase in serum interleukin-6 caused by RA induction to levels observed in non-arthritic animals. In addition, BVA therapy significantly reduced arthritis-induced nociceptive behaviors (i.e. nociceptive scores for mechanical hyperalgesia and thermal hyperalgesia). Finally, BVA treatment significantly suppressed adjuvant-induced Fos expression in the lumbar spinal cord at 3 weeks post-adjuvant injection. In contrast, BVE treatment (0.05 mg/kg/day) failed to show any anti-inflammatory or antinociceptive effects on RA. The results of the present study demonstrate that BVA is the effective fraction of whole BV responsible for the antinociception and anti-inflammatory effects of BV acupuncture treatment. Thus it is recommended that this fraction of BV be used for long-term treatment of RA-induced pain and inflammation. However, further study is necessary to clarify which constituents of the BVA fraction are directly responsible for these anti-arthritis effects.

Intrathecal injection of the ς1 receptor antagonist BD1047 blocks both mechanical allodynia and increases in spinal NR1 expression during the induction phase of rodent neuropathic pain

Background: Selective blockade of spinal &sfgr;1 receptors (Sig-1R) suppresses nociceptive behaviors in the mouse formalin test. The current study was designed to verify whether intrathecal Sig-1R antagonists can also suppress chronic neuropathic pain. Methods: Neuropathic pain was produced by chronic constriction injury (CCI) of the right sciatic nerve in rats. The Sig-1R antagonist BD1047 was administered intrathecally twice daily from postoperative days 0 to 5 (induction phase of neuropathic pain) or from days 15 to 20 (maintenance phase). Western blot and immunohistochemistry were performed to determine changes in Sig-1R expression and to examine the effect of BD1047 on N-methyl-d-aspartate receptor subunit 1 expression and phosphorylation in spinal cord dorsal horn from neuropathic rats. Results: BD1047 administered on postoperative days 0–5 significantly attenuated CCI-induced mechanical allodynia, but not thermal hyperalgesia, and this suppression was blocked by intrathecal administration of the Sig-1R agonist PRE084. In contrast, BD1047 treatment during the maintenance phase of neuropathic pain had no effect on mechanical allodynia. Sig-1R expression significantly increased in the ipsilateral spinal cord dorsal horn from days 1 to 3 after CCI. Importantly, BD1047 (30 nmol) administered intrathecally during the induction, but not the maintenance phase, blocked the CCI-induced increase in N-methyl-d-aspartate receptor subunit 1 expression and phosphorylation. Conclusions: These results demonstrate that spinal Sig-1Rs play a critical role in both the induction of mechanical allodynia and the activation of spinal N-methyl-d-aspartate receptors in CCI rats and suggest a potential therapeutic role for the use of Sig-1R antagonists in the clinical management of neuropathic pain.

The analgesic efficacy of bee venom acupuncture for knee osteoarthritis: a comparative study with needle acupuncture.

The aim of this investigation was to determine whether bee venom (BV) administered directly into an acupoint was a clinically effective and safe method for relieving the pain of patients with knee osteoarthritis (OA) as compared to traditional needle acupuncture. We evaluated the efficacy of BV acupuncture using both pain relief scores and computerized infrared thermography (IRT) following 4 weeks of BV acupuncture treatment. We observed that a significantly higher proportion of subjects receiving BV acupuncture reported substantial pain relief as compared with those receiving traditional needle acupuncture therapy. Furthermore, the IRT score was significantly improved and paralleled the level of pain relief.

Sonic hedgehog stimulates mouse embryonic stem cell proliferation by cooperation of Ca2+/protein kinase C and epidermal growth factor receptor as well as Gli1 activation.

Hedgehog signaling has an essential role in the control of stem cell growth in embryonic tissues. Therefore, this study examined the effect of sonic hedgehog (Shh) on the self‐renewal of mouse embryonic stem (ES) cells and its related mechanisms. Shh increased DNA synthesis blocked by the inhibition of the smoothened receptor. Shh required Gli1 activation to induce the increases in Notch/Hes‐1 and Wnt/β‐catenin. Shh increased the intracellular calcium concentration ([Ca2+]i) and protein kinase C (PKC) activity. We show that the Shh‐induced increase in the Gli1 mRNA level requires [Ca2+]i and PKC. Shh increased the phosphorylation of epidermal growth factor receptor (EGFR), which is blocked by the matrix metalloproteinase inhibitor. Subsequently, Shh increased the nuclear factor (NF)‐κB p65 phosphorylation, which was inhibited by blocking PKC and EGFR tyrosine kinase. Shh also increased the level of the cell cycle regulatory proteins in a dose‐dependent manner. However, Shh decreased the levels of the cyclin‐dependent kinase inhibitory proteins. The effect of Shh on these proteins was inhibited by blocking PKC, EGFR, and NF‐κB as well as transfection of Gli1 small interfering RNA (siRNA). Finally, Shh‐induced progression of the G1/S‐phase was blocked by the inhibition of PKC and EGFR tyrosine kinase. In conclusion, Shh stimulates mouse ES cell proliferation through Gli1 activation as well as Ca2+/PKC and EGFR.

High glucose regulates cyclin D1/E of human mesenchymal stem cells through TGF-beta1 expression via Ca2+/PKC/MAPKs and PI3K/Akt/mTOR signal pathways.

The elucidation of factors that support human mesenchymal stem cells (hMSCs) growth has remained unresolved partly because of the reliance of many researchers on ill‐defined, proprietary medium formulation. Thus, we investigated the effects of high glucose (D‐glucose, 25 mM) on hMSCs proliferation. High glucose significantly increased [3H]‐thymidine incorporation and cell‐cycle regulatory protein expression levels compared with 5 mM D‐glucose or 25 mM L‐glucose. In addition, high glucose increased transforming growth factor‐β1 (TGF‐β1) mRNA and protein expression levels. High glucose‐induced cell‐cycle regulatory protein expression levels and [3H]‐thymidine incorporation, which were inhibited by TGF‐β1 siRNA transfection and TGF‐β1 neutralizing antibody treatment. High glucose‐induced phosphorylation of protein kinase C (PKC), p44/42 mitogen‐activated protein kinases (MAPKs), p38 MAPK, Akt, and mammalian target of rapamycin (mTOR) in a time‐dependent manner. Pretreatment of PKC inhibitors (staurosporine, 10−6 M; bisindolylmaleimide I, 10−6 M), LY 294002 (PI3 kinase inhibitor, 10−6 M), Akt inhibitor (10−5 M), PD 98059 (p44/42 MAPKs inhibitor, 10−5 M), SB 203580 (p38 MAPK inhibitor, 10−6 M), and rapamycin (mTOR inhibitor, 10−8 M) blocked the high glucose‐induced cellular proliferation and TGF‐β1 protein expression. In conclusion, high glucose stimulated hMSCs proliferation through TGF‐β1 expression via Ca2+/PKC/MAPKs as well as PI3K/Akt/mTOR signal pathways. J. Cell. Physiol. 224:59–70, 2010

Functional recovery after spinal cord injury in dogs treated with a combination of Matrigel and neural-induced adipose-derived mesenchymal Stem cells.

BACKGROUND AIMS Previous studies have reported that scaffold or cell-based transplantation may improve functional recovery following spinal cord injury (SCI), but these results were based on neuronal regeneration and cell replacement. In this study, we investigated whether a combination of Matrigel and neural-induced mesenchymal stem cells (NMSC) improved hindlimb function in dogs with SCI, and what mechanisms were involved. METHODS We pre-differentiated canine adipose-derived mesenchymal stem cells into NMSC. A total of 12 dogs subjected to SCI procedures were assigned to one of the following three transplantation treatment groups: phosphate-buffered saline (PBS); Matrigel; or Matrigel seeded with NMSC. Treatment occurred 1 week after SCI. Basso, Beattie and Bresnahan (B.B.B.) and Tarlov scores, histopathology, immunofluorescence staining and Western blot analysis were used to evaluate the treatment effects. RESULTS Compared with dogs administered PBS or Matrigel alone, dogs treated with Matrigel + NMSC showed significantly better functional recovery 8 weeks after transplantation. Histology and immunochemical analysis revealed that the combination of Matrigel + NMSC reduced fibrosis from secondary injury processes and improved neuronal regeneration more than the other treatments. In addition, the combination of Matrigel + NMSC decreased the expression of inflammation and/or astrogliosis markers. Increased expressions of intracellular molecules related to neuronal extension, neuronal markers and neurotrophic factors were also found in the Matrigel + NMSC group. However, the expression of nestin as a neural stem cell marker was increased with Matrigel alone. CONCLUSIONS The combination of Matrigel + NMSC produced beneficial effects in dogs with regard to functional recovery following SCI through enhancement of anti-inflammation, anti-astrogliosis, neuronal extension and neuronal regeneration effects.

Visceral antinociception produced by bee venom stimulation of the Zhongwan acupuncture point in mice: role of α2 adrenoceptors

The goal of the present study was to determine whether bee venom (BV) injection into the Zhongwan acupoint (CV12), compared to injection into a non-acupoint, produced antinociception in an acetic acid-induced visceral pain model. This was accomplished by injecting BV subcutaneously into the Zhongwan acupoint or into a non-acupoint 30 min before intraperitoneal injection of acetic acid in ICR mice. BV injection into the acupoint produced a dose dependent suppression of acetic acid-induced abdominal stretches and of acetic acid-induced Fos expression in the spinal cord and the nucleus tractus solitarii. In contrast BV injection into the non-acupoint only produced antinociception at the highest dose of BV tested. Naloxone pretreatment did not alter the antinociceptive effect of BV acupoint injection on the abdominal stretch reflex. On the other hand, pretreatment with the alpha 2-adrenoceptor antagonist, yohimbine completely blocked the antinociceptive effect of BV acupoint injection. These results imply that BV acupoint stimulation can produce visceral antinociception that is associated with activation of alpha 2-adrenoceptors, but not with naloxone-sensitive opioid receptors.

Collagen I regulates the self-renewal of mouse embryonic stem cells through α2β1 integrin- and DDR1-dependent Bmi-1.

Adhesion of cells to extracellular matrix (ECM) influences vital aspects of anchorage‐dependent cell behavior including survival, proliferation, and differentiation. However, the role of collagen I in mouse embryonic stem cells (mESCs) is not well‐known. Therefore, in the present study we examined the effect of collagen I on mESC self‐renewal and related signal pathways. Collagen I (10 µg/ml) maintained mESCs in an undifferentiated state (Nanog, OCT4, and SSEA‐1) and did not affect differentiation (GATA4, Tbx5, Fgf5, and Cdx2) in the presence of leukemia inhibitory factor (LIF). Treatment with collagen I bound both α2β1 integrin and discoidin domain receptor 1 (DDR1), and stimulated intracellular signaling pathways. Collagen I‐bound α2β1 integrin increased integrin‐linked kinase (ILK) phosphorylation, cleaved Notch protein expression in the nuclear fraction, and Gli‐1 mRNA expression. In addition, collagen I‐bound DDR1 increased GTP‐bound Ras, phosphoinositide 3‐kinase (PI3K) p85α catalytic subunit protein expression, and Akt and ERK phosphorylation. Importantly, collagen I increased Bmi‐1 protein expression in the nucleus which was blocked by small interfering RNA (siRNA) specific for Gli‐1 and ERK, showing that parallel pathways of integrins and DDR1 merge at Bmi‐1. Furthermore, collagen I‐induced p16 decrease and p‐Rb increase were reversed by Bmi‐1‐specific siRNA. Moreover, Bmi‐1 silencing abolished the collagen I‐induced increase of proliferation indices and undifferentiation markers. These results indicate that collagen I stimulates the self‐renewal of mESCs mediated by Bmi‐1 through α2β1 integrin‐dependent ILK, Notch, Gli‐1, and DDR1‐dependent Ras, PI3K/Akt, and ERK. J. Cell. Physiol. 226: 3422–3432, 2011.

Involvement of caveolin-1 in fibronectin-induced mouse embryonic stem cell proliferation: role of FAK, RhoA, PI3K/Akt, and ERK 1/2 pathways.

Fibronectin (FN) is the foremost proliferation‐associated extracellular matrix component promoting cell adhesion, migration, and survival. We examined the effect of FN on cell proliferation and the related signaling pathways in mouse embryonic stem (ES) cells. FN increased integrin β1, Src, focal adhesion kinase (FAK), and caveolin‐1 phosphorylation levels in a time‐dependent manner. Phosphorylation of Src, FAK, and caveolin‐1 was attenuated by integrin β1 neutralizing antibody. Integrin β1, Src, and FAK coimmunoprecipitated with caveolin‐1 in the presence of FN. In addition, FN increased RhoA and Rho kinase activation, which were completely blocked by PP2, FAK small interfering RNA (siRNA), caveolin‐1 siRNA, or the caveolar disruptor methyl‐β‐cyclodextrin (MβCD). FN also increased phosphorylation of Akt and ERK 1/2, which were significantly blocked by either FAK siRNA, caveolin‐1 siRNA, MβCD, GGTI‐286 (RhoA inhibitor), or Y‐27632 (Rho kinase inhibitor). FN‐induced increase of protooncogenes (c‐fos, c‐myc, and c‐Jun) and cell‐cycle regulatory proteins (cyclin D1/CDK4 and cyclin E/CDK2) expression levels were attenuated by FAK siRNA or caveolin‐1 siRNA. Furthermore, inhibition of each pathway such as integrin β1, Src, FAK, caveolin‐1, RhoA, Akt, and ERK 1/2 blocked FN‐induced [3H]‐thymidine incorporation. We conclude that FN stimulates mouse ES cell proliferation via RhoA‐PI3K/Akt‐ERK 1/2 pathway through caveolin‐1 phosphorylation. J. Cell. Physiol. 226: 267–275, 2010.