E.S.K. Ng

Mechanisms of the relaxant effect of a Danshen and Gegen formulation on rat isolated cerebral basilar artery

AIM OF THE STUDY Danshen (root of Salvia miltiorrhiza) and gegen (root of Pueraria lobata) are two herbs used in traditional Chinese medicine, most commonly for their putative cardioprotective and anti-atherosclerotic effects. In this study, the actions of a danshen and gegen formulation (DG; ratio 7:3) were investigated on rat-isolated cerebral basilar artery. MATERIALS AND METHODS Rat basilar artery rings were precontracted with 100 nM U46619. Involvement of endothelium-dependent mechanisms was investigated by mechanical removal of the endothelium; K(+) channels were investigated by pretreatment of the artery rings with various K(+) channel inhibitors, and Ca(2+) channels were investigated in artery rings incubated with Ca(2+)-free buffer and primed with 100 nM U46619 for 5 min prior to adding CaCl(2) to elicit contraction. RESULTS DG produced concentration-dependent relaxation of the artery rings with an IC(50) of 895±121 μg/ml. Mechanical removal of the endothelium or pretreatment with the BK(Ca) channel inhibitor iberiotoxin (100 nM), the K(V) channel inhibitor 4-aminopyridine (1 mM), or the K(IR) channel inhibitor barium chloride (100 μM), all had no effect on the DG-induced response (P>0.05 for all). However, pretreatment with the K(ATP) channel inhibitor glibenclamide (1 μM), the non-selective K(+) channel inhibitor tetraethylammonium (TEA, 100 mM), or a combination of all the K(+) channel inhibitors (iberiotoxin+4-aminopyrindine+barium chloride+glibenclamide+TEA) produced significant inhibition on the DG-induced response (P<0.01 for all); its maximum vasorelaxant effect (Imax) was reduced by 37, 24, and 30%, respectively. Preincubation of the artery rings with DG for 10 min produced concentration-dependent (1, 3 and 7 mg/ml) and total inhibition on the CaCl(2)-induced vasoconstriction. CONCLUSIONS These findings suggest the vasorelaxant effect of DG on rat basilar artery is independent of endothelium-derived mediators, whereas, inhibition of Ca(2+) influx in the vascular smooth muscle cells is important, and a minor component is mediated by the opening of K(ATP) channels. DG could be a useful cerebroprotective agent in some patients with occlusive cerebrovascular disease.

Analgesic and anti-arthritic effects of Lingzhi and San Miao San supplementation in a rat model of arthritis induced by Freund's complete adjuvant.

AIM OF STUDY In this study, we have investigated the analgesic and anti-arthritic effects of a traditional Chinese medicine (TCM) combination of Lingzhi and San Miao San (SMS) in a rat model of arthritis induced by Freunds complete adjuvant (FCA). MATERIALS AND METHODS Sprague-Dawley rats were induced with monoarthritis by single unilateral injection of FCA into the knee joint. The TCM combination was administered to the rats daily by intraperitoneal injection (50mg/(kgday)) or via oral administration (500mg/(kgday)) for 7 days before induction of arthritis and 7 days after. Extension angle that provoked struggling behavior, and size and blood flow of the rat knees were measured to give indexes of allodynia, edema, and hyperemia, respectively. The extent of cell infiltration, tissue proliferation, and erosions of joint cartilage provided additional indexes of the arthritis condition. RESULTS FCA injection produced significant allodynia, edema, hyperemia, immune cell infiltration, synovial tissue proliferation, and erosions of joint cartilage in the ipsilateral knees compared with the contralateral saline-injected knees. Intraperitoneal injection of the TCM combination (50mg/(kgday)) suppressed allodynia, edema, and hyperemia in the inflamed knees, and oral administration (500mg/(kgday)) suppressed edema and hyperemia. Histological examination showed that the TCM administered by either route reduced immune cell infiltration and erosion of joint cartilage. CONCLUSIONS These findings suggest the Lingzhi and SMS formulation has analgesic and anti-inflammatory effects in arthritic rat knees, and concur to previous clinical studies that showed the TCM combination reduced pain in rheumatoid arthritis patients, and extends its possible benefit to suppression of inflammatory symptoms in these patients.

Mechanisms of the cerebral vasodilator actions of isoflavonoids of Gegen on rat isolated basilar artery.

ETHNOPHARMACOLOGICAL RELEVANCE Gegen (root of Pueraria lobata) is used in traditional Chinese medicine for treatment of cardiovascular diseases. In this study, the relaxant actions of three of its isoflavonoids; puerarin, daidzein, and daidzin, were investigated on rat-isolated cerebral basilar artery. MATERIALS AND METHODS Rat basilar artery rings were precontracted with 100 nM U46619. Involvement of endothelium-dependent mechanisms was investigated by mechanical removal of the endothelium and inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX) enzymes. Adenylyl cyclase- and guanylyl cyclase-dependent pathways were investigated using their respective inhibitors 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536) and 1H-[1,2,4]oxadiazolo [4,3-[alpha]]-quinoxalin-1-one (ODQ). K(+) channels were investigated by pretreatment of the artery rings with various K(+) channel inhibitors, and Ca(2+) channels were investigated in artery rings incubated with Ca(2+)-free buffer and primed with 100 nM U46619 for 5 min prior to adding CaCl(2) to elicit contraction. RESULTS Puerarin, daidzein, and daidzin produced concentration-dependent relaxation of the artery rings with concentration that produced 50% inhibition (IC(50)) of 304 ± 49 μM, 20 ± 7 μM, and 140 ± 21 μM, respectively. Removal of the endothelium produced no change on their vasorelaxant responses except the maximum response (I(max)) to puerarin was inhibited by 28%. The NOS inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME; 100 μM) also produced 45% inhibition on the puerarin-induced vasorelaxant response, but not the COX inhibitor flurbiprofen (10 μM). SQ22536 (100 μM) and ODQ (100μM) did not affect the vasodilator responses to puerarin, daidzein and daidzin, but glibenclamide (1μM), tetraethylammonium (TEA, 100mM) or a combination of K(+) channel inhibitors (100nM iberiotoxin+1mM 4-aminopyridine+100 μM barium chloride+1 μM glibenclamide+100mM TEA) reduced their I(max). The contractile response to CaCl(2) was attenuated by 61% and 34% in the presence of daidzein and daidzin, respectively, whereas, puerarin did not significantly affect the contraction. CONCLUSIONS The vasorelaxant action of daidzein and daidzin involved opening of K(+) channels and inhibition of Ca(2+) influx in the vascular smooth muscle cells. There is no evidence supporting involvement of endothelium-derived relaxing factors (EDRFs) in their actions. In contrast, puerarin produced vasodilatation via an endothelium-dependent mechanism involving nitric oxide production and an endothelium-independent pathway mediated by the opening of K(+) channels. The cerebral vasodilator activities of all these three isoflavonoids may be beneficial to patients with obstructive cerebrovascular diseases.

Reversal of endothelial progenitor cell dysfunction in patients with type 2 diabetes using a conditioned medium of human embryonic stem cell-derived endothelial cells

The potential clinical application of bone marrow or peripheral blood‐derived progenitor cells for cardiovascular regeneration in patients with diabetes mellitus (DM) is limited by their functional impairment. We sought to determine the mechanisms of impaired therapeutic efficacy of peripheral blood‐derived progenitor cells in type 2 DM patients and evaluated the use of cell‐free conditioned medium obtained from human embryonic stem cell‐derived endothelial‐like cells (ESC‐ECs) to reverse their functional impairment.

The anti-arthritic effects of Aconitum vilmorinianum, a folk herbal medicine in Southwestern China.

ETHNOPHARMACOLOGICAL RELEVANCE Aconiti Radix (AC) and Aconiti Kusnezoffii Radix (AK) are two traditional Chinese medicines commonly used to treat joint pain and arthritis. In Southwestern China, Huangcaowu (AV), the root of Aconitum vilmorinianum Kom., has long been used as a local substitute for these herbs for analgesia and anti-inflammation. However, its anti-arthritic effects have not been investigated. AIM OF STUDY To investigate the anti-arthritic effects of Huangcaowu (AV). MATERIALS AND METHODS Mono-arthritis in SD rats was induced by unilateral intra-articular injection of Freunds complete adjuvant. Physiological saline was injected in the contralateral knee. Seventy five percent ethanol extracts of AV (10 mg/kg/day and 100 mg/kg/day), AC (100 mg/kg/day) and AK (100 mg/kg/day) were administered to rats by oral gavage for 14 consecutive days (Day -6 to Day 7) while arthritis was induced at the seventh day (Day 0). The anti-arthritic effects of the herbs were assessed by measuring allodynia, swelling, hyperaemia and the vascular permeability of the knee joints. RESULTS AV (10 mg/kg/day and 100 mg/kg/day), AC (100 mg/kg/day) and AK (100 mg/kg/day) suppressed joint allodynia. AV (10 mg/kg/day and 100 mg/kg/day) and AK (100 mg/kg/day) significantly reduced join swelling and hyperaemia while AC (100 mg/kg/day) did not. AV (100 mg/kg/day) attenuated vascular permeability while AC (100 mg/kg/day) and AK (100 mg/kg/day) showed no improvement. CONCLUSIONS Huangcaowu (AV) significantly improved allodynia, swelling, hyperaemia and vascular permeability in arthritic knee joints. It showed the highest anti-arthritic effects among the three tested Aconitum herbs.

Cerebral vasodilator properties of Danshen and Gegen: A study of their combined efficacy and mechanisms of actions

Danshen and Gegen are two commonly used Chinese herbal medicines for treatment of cardiovascular diseases. The aim of the present study was to elucidate the combination effects of these two herbs on cerebral vascular tone and their underlying mechanisms of actions. Basilar artery rings were obtained from rats and precontracted with U46619. Cumulative administrations of aqueous extracts of Danshen, Gegen, or the two herbs combined (DG; ratio 7:3) produced concentration-dependent relaxation of the artery rings. Statistical analysis on these findings produced a combination index (CI) of 1.041 at ED50, which indicates the two herbs produced additive vasodilator effects when used as a combined decoction. Removal of the endothelium had no effect on the vasodilator properties of Danshen, Gegen, and DG. However, their maximum effects (Imax) were significantly blunted by a KATP channel inhibitor glibenclamide, a non-selective K(+) channel inhibitor tetraethylammonium (TEA), and by a combination of K(+) channel inhibitors (glibenclamide+TEA+iberiotoxin+4-aminopyridine+barium chloride). In addition, Danshen, Gegen, and DG produced augmentation of KATP currents and inhibited Ca(2+) influx in vascular smooth muscle cells isolated from rat basilar arteries. Furthermore, these agents inhibited CaCl2-induced contraction in the artery rings. In conclusion, the present study showed that Danshen and Gegen produced additive vasodilator effects on rat cerebral basilar arteries. These effects were independent of endothelium-derived relaxant factors (EDRF), but required the opening of KATP channels and inhibition of Ca(2+) influx in the vascular smooth muscle cells. It is suspected that the cerebral vasodilator effects of Danshen and Gegen produced either on their own or in combination, can help patients with obstructive cerebrovascular diseases.

Characterisation of somatostatin actions on knee joint blood vessels of the rat.

The effects of somatostatin on blood flow, plasma extravasation and knee joint sizes in the rat were investigated. Topical bolus administrations of somatostatin (10 pmol-100 nmol) onto the exposed rat knee joint capsules produced dose-dependent increases in knee joint blood flow with an ED(50) value of 1.7 nmol, and a maximum increase of 109.7%. The peak vasodilator response was observed at 1 min following drug administration, and it subsided at 5 min. Treatment of the rat knee with a somatostatin receptor antagonist cyclo(7-aminoheptanoul-Phe-D-Trp-Lys-Thr[Bzl] (cyclo-somatostatin; 2 x 20 nmol) significantly suppressed the somatostatin-induced vasodilator response, but treatment with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 2 x 50 nmol) or the cyclo-oxygenase inhibitor flurbiprofen (2 x 10 nmol) had no effect. Unilateral intraarticular injections of somatostatin (10 nmol) produced no change on blood flow and sizes of the rat knee joints, but elicited marked ipsilateral Evans blue extravasation. Cyclo-somatostatin at doses of 2 x 20 and 2 x 50 nmol did not affect the plasma extravasation response to somatostatin. The present findings indicate the vasodilator effect of somatostatin is mediated by receptors sensitive to cyclo-somatostatin inhibition, but its plasma extravasation effect might be mediated by somatostatin receptor types that are resistant to inhibition by cyclo-somatostatin. There is no evidence that nitric oxide and prostaglandins are involved in the somatostatin-induced vasodilator response. It is suspected that the vascular effects of somatostatin demonstrated in this study would play a part in the innate response of an inflammatory reaction.

Predicting the hourly Hong Kong representative sky from Typical Meteorological Year data for dynamic daylighting simulation

This paper proposes a method to predict the hourly Hong Kong representative sky from Typical Meteorological Year data. An evaluation of the sensitivity of the vertical sky component demonstrates that the Hong Kong representative sky can provide better results than the Perez all-weather sky model. Error reductions of 9–23% can be expected in the four cardinal directions. To predict the Hong Kong representative sky from the Typical Meteorological Year data, a clear sky index can be derived from global horizontal irradiance. Three Hong Kong representative skies can be classified by the clear sky index corresponding to skies 1, 8 and 13 of the Hong Kong representative sky, respectively. The predicted hourly Hong Kong representative sky can provide the sky luminance data for the dynamic simulation of buildings.

The characterization of a full-thickness excision open foot wound model in n5-streptozotocin (STZ)-induced type 2 diabetic rats that mimics diabetic foot ulcer in terms of reduced blood circulation, higher C-reactive protein, elevated inflammation, and reduced cell proliferation

Delayed foot wound healing is a major complication attributed to hyperglycemia in type 2 diabetes mellitus (DM) patients, and these wounds may develop into foot ulcers. There are at least two types of DM wound models used in rodents to study delayed wound healing. However, clinically relevant animal models are not common. Most models use type 1 DM rodents or wounds created on the back rather than on the foot. An open full-thickness excision wound on the footpad of type 2 DM rats is more clinically relevant, but such a model has not yet been characterized systematically. The objective of this study was to investigate and characterize how DM affected a full-thickness excision open foot wound in n5-streptozotocin (n5-STZ)-induced type 2 DM rats. We hypothesized that elevated inflammation, reduced blood circulation, and cell proliferation due to hyperglycemia could delay the wound healing of DM rats. The wounds of DM rats were compared with those of non-DM rats (Ctrl) at Days 1 and 8 post wounding. The wound healing process of the DM rats was significantly delayed compared with that of the Ctrl rats. The DM rats also had higher C-reactive protein (CRP) and lower blood circulation and proliferating cell nuclear antigen (PCNA) in DM wounds. This confirmed that elevated inflammation and reduced blood flow and cell proliferation delayed foot wound healing in the n5-STZ rats. Hence, this open foot wound animal model provides a good approach to study the process of delayed wound healing.

Suppression of ischaemia-induced injuries in rat brain by protease-activated receptor-1 (PAR-1) activating peptide.

Ischaemic stroke has become one of the leading causes of death and disability worldwide. The role of protease activated receptor-1 (PAR-1) in this disease is uncertain. In the present study, the actions of a protease activated receptor-1 activating peptide (PAR-1 AP) SFLLRN-NH2 were investigated in an in vivo rat model of ischaemic stroke induced by middle cerebral artery occlusion (MCAO) and in an in vitro model induced by oxygen and glucose deprivation (OGD) in primary cultured rat embryonic cortical neurones. Rats subjected to MCAO exhibited increased brain infarct volume, oedema, and neurological deficit. Rat cortical neurones subjected to OGD showed increased lactate dehydrogenase, caspase-3 activity and TUNEL positive cells, whereas, mitochondrial membrane potential and cell viability were decreased. Furthermore, both models had elevated levels of reactive oxygen species, nitrite, and malondialdehyde, while anti-oxidant enzymes and bcl-2/bax ratio were decreased. These detrimental changes were suppressed by SFLLRN-NH2, and its protective actions were inhibited by a PAR-1 antagonist (BMS-200261). In summary, SFLLRN-NH2 was found to possess anti-oxidant and anti-apoptotic properties, and it produced marked inhibition on the detrimental effects of ischaemia in in vivo and in vitro models of ischaemic stroke. The present findings suggest PAR-1 is a promising target for development of novel treatments of ischaemic brain disease.