Flavonoid Myricetin Modulates GABA(A) Receptor Activity through Activation of Ca(2+) Channels and CaMK-II Pathway.

The flavonoid myricetin is found in several sedative herbs, for example, the St. John's Wort, but its influence on sedation and its possible mechanism of action are unknown. Using patch-clamp technique on a brain slice preparation, the present study found that myricetin promoted GABAergic activity in the neurons of hypothalamic paraventricular nucleus (PVN) by increasing the decay time and frequency of the inhibitory currents mediated by GABA(A) receptor. This effect of myricetin was not blocked by the GABA(A) receptor benzodiazepine- (BZ-) binding site antagonist flumazenil, but by KN-62, a specific inhibitor of the Ca(2+)/calmodulin-stimulated protein kinase II (CaMK-II). Patch clamp and live Ca(2+) imaging studies found that myricetin could increase Ca(2+) current and intracellular Ca(2+) concentration, respectively, via T- and L-type Ca(2+) channels in rat PVN neurons and hypothalamic primary culture neurons. Immunofluorescence staining showed increased phosphorylation of CaMK-II after myricetin incubation in primary culture of rat hypothalamic neurons, and the myricetin-induced CaMK-II phosphorylation was further confirmed by Western blotting in PC-12 cells. The present results suggest that myricetin enhances GABA(A) receptor activity via calcium channel/CaMK-II dependent mechanism, which is distinctively different from that of most existing BZ-binding site agonists of GABA(A) receptor.

Bak Foong Pills induce an analgesic effect by inhibiting nociception via the somatostatin pathway in mice.

Dysmenorrhoea, defined as cramping pain in the lower abdomen occurring before or during menstruation, affects, to varying degrees, up to 90% of women of child-bearing age. We investigated whether BFP (Bak Foong Pills), a traditional Chinese medicine treatment for dysmenorrhoea, possesses analgesic properties. Results showed that BFP was able to significantly reduce pain responses following subchronic treatment for 3 days, but not following acute (1 h) treatment in response to acetic acid-induced writhing in C57/B6 mice. The analgesic effect was not due to inhibition of COX (cyclo-oxygenase) activity, evidenced by the lack of inhibition of prostacyclin and PGE2 (prostaglandin E2) production. Molecular analysis revealed that BFP treatment modulated the expression of a number of genes in the spinal cord of mice subjected to acetic acid writhing. RT-PCR (reverse transcription-PCR) analysis of spinal cord samples showed that both sst4 (somatostatin receptor 4) and sst2 receptor mRNA, but not µOR (µ-opiate receptor) and NK1 (neurokinin-1) receptor mRNA, were down-regulated following BFP treatment, thus implicating somatostatin involvement in BFP-induced analgesia. Administration of c-som (cyclo-somatostatin), a somatostatin antagonist, prior to acetic acid-induced writhing inhibited the analgesic effect. Thus subchronic treatment with BFP has anti-nociceptive qualities mediated via the somatostatin pathway.

Bak Foong protects dopaminergic neurons against MPTP-induced neurotoxicity by its anti-apoptotic activity.

Bak Foong pill (BFP) is a well-known traditional Chinese medicine used for treatment of various gynaecological disorders. In addition, it exerts beneficial effects on other functional systems including the central nervous system. In the present study, we have investigated the possible neuroprotective action of BFP upon the nigrostriatal dopaminergic system by examining its effect on the expression patterns of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the 1-methyl-4-phenyl-1,2,3,6-tetrahyrdropyridine (MPTP)-induced Parkinson's disease (PD) mouse model. MPTP significantly decreased TH and DAT mRNA levels in the striatum and midbrain of both female and male C57BL/6 mice. However, with BFP pre-treatment mice showed a reduced neurotoxicity, with TH and DAT mRNA levels either not affected by MPTP or affected to a lesser extent in the midbrain and striatum when compared to vehicle treated animals. Possible anti-apoptotic activity of BFP was further studied in a dopamine-secreting neuroendocrine cell line, PC12. In this assay, MPTP elevated the expression of a pro-apoptotic gene, Bax, while this expression was reduced by BFP pre-treatment. Flow cytometry results also revealed that the effect of MPTP-induced apoptosis in PC12 cell lines was significantly reduced by BFP. The present results suggest that BFP is able to protect dopaminergic neurons from neurotoxin-induced neuronal injury with anti-apoptotic activity being one of the possible mechanisms.

Improvement of barrier function and stimulation of colonic epithelial anion secretion by Menoease Pills.

AIM: Menoease Pills (MP), a Chinese medicine-based new formula for postmenopausal women, has been shown to modulate the endocrine and immune systems. The present study investigated the effects of MP and one of its active ingredients, ligustrazine, on epithelial barrier and ion transport function in a human colonic cell line, T84. METHODS: Colonic transepithelial electrophysiological characteristics and colonic anion secretion were studied using the short circuit current (ISC) technique. RT-PCR was used to examine the expression of cytoplasmic proteins associated with the tight junctions, ZO-1 (zonula occludens-1) and ZO-2 (zonula occludens-2). RESULTS: Pretreatment of T84 cells with MP (15 microg/mL) for 72 h significantly increased basal potential difference, transepithelial resistance and basal ISC. RT-PCR results showed that the expressions of ZO-1 and ZO-2 were significantly increased after MP treatment, consistent with improved epithelial barrier function. Results of acute stimulation showed that apical addition of MP produced a concentration-dependent (10-5,000 microg/mL, EC50 = 293.9 microg/mL) increase in ISC. MP-induced ISC was inhibited by basolateral treatment with bumetanide (100 micromol/L), an inhibitor of the Na+-K+-2Cl- cotransporter, apical addition of Cl- channel blockers, diphenylamine-2, 2'-dicarboxylic acid (1 mmol/L) or glibenclamide (1 mmol/L), but not 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid or epithelial Na+ channel blocker, amiloride. The effect of MP on ZO-1 and ZO-2 was mimicked by Ligustrazine and the ligustrazine-induced ISC was also blocked by basolateral application of bumetanide and apical addition of diphenylamine-2, 2'-dicarboxylic acid or glibenclamide, and reduced by a removal of extracellular Cl-. CONCLUSION: The results of the present study suggest that MP and lligustrazine may improve epithelial barrier function and exert a stimulatory effect on colonic anion secretion, indicating the potential use of MP and its active ingredients for improvement of GI tract host defense and alleviation of constipation often seen in the elderly.

Cardiovascular protective effects of traditional Chinese medicine bak foong pills in spontaneously hypertensive rats.

The cardiovascular protective effects of the traditional Chinese medicine Bak Foong Pills (BFP) were investigated. Spontaneously hypertensive rats (SHR) were treated (3 g/kg) over a 5-month period and blood pressure measurements periodically tested with a plethysmographic tail cuff. Following treatment, blood samples were analysed for serum electrolyte levels and lipid levels and brain tissue subjected to micro-array analysis. In vitro experiments were also conducted to identify possible direct vasorelaxatory effect. The results showed that BFP was able to significantly reduce both systolic and diastolic blood pressure by about 30 mmHg in SHR following 5 months of treatment, when compared to untreated animals. Investigation for possible mechanisms of actions revealed that BFP treated rats had elevated blood serum K(+) levels, and also demonstrated decreased serum triglyceride levels. Micro-array analysis of brain tissue showed altered expression of acetylcholine and lysosphingolipid receptor genes that are known to regulate blood pressure. In vitro experiments also showed that BFP caused a concentration-dependant vasorelaxation of isolated rat aortae when contracted with phenylepherine, which was partially inhibited by nitric oxide synthase inhibitor L-NAME (100 microM). These data suggest that BFP is able to significantly reduce hypertension in SHR through mechanisms probably involving a combination of increased serum K(+), vasorelaxatory action, reduced serum triglyceride and altered gene regulation in the higher centres.