Yu Lin Gou

Plasticity of rat bone marrow-derived 5-hydroxytryptamine-sensitive neurons: dedifferentiation and redifferentiation

Inducing cellular dedifferentiation has been proposed as a potential method for enhancing endogenous regeneration in mammals. Here we demonstrate that phenotypic and functional neurons derived from adult rat bone marrow stromal stem cells (MSCs) can be induced to undergo dedifferentiation, then proliferation and redifferentiation. In addition to morphological changes and expression of neuronal markers, neuron‐specific enolase and neurofilament H, functional differentiation was monitored by intracellular Ca2+ mobilization in response to a ubiquitous neurotransmitter, 5‐hydroxytryptamine (5‐HT) at different stages. The neurons derived from rMSCs were found to have increased 5‐HT response. This 5‐HT sensitivity could be reversed to basal level similar to that found in rMSCs when neurons, up to 3 days after neuronal induction, were induced to undergo dedifferentiation. Increase in 5‐HT‐induced Ca2+ mobilization was again observed when rMSCs derived from dedifferentiated neurons were induced to redifferentiate into neurons again. Variation in 5‐HT1A receptor immunoreactivity was observed in stem cells, differentiated neurons, dedifferentiated neurons and redifferentiation neurons, consistent with their respective 5‐HT sensitivity. These results suggest that adult bone marrow‐derived 5‐HT sensitive neurons are capable of dedifferentiation, then proliferation and redifferentiation, indicating their plasticity and potential use in treatment of neural degenerative diseases.

Anti‐apoptotic activity of Bak Foong Pills and its ingredients on 6‐hydroxydopamine‐induced neurotoxicity in PC12 cells

Bak Foong Pills (BFP), a traditional Chinese medicine used for centuries for the enhancement of womens health, was shown to display neuro‐protective activity in the 1‐methyl‐4‐phenyl‐1,2,4,6,‐tetrahydro‐pyridine (MPTP)‐induced mouse model in a previous study. In order to elucidate its mechanism of action, we investigated the anti‐apoptotic properties of Bak Foong Pills and its main ingredients, including Panax ginseng, Angelica sinensis, Glycyrrhiza uralensis, and Ligusticum chuanxiong, in the 6‐hydroxydopamine (6‐OHDA)‐treated PC12 cell model. The addition of the neurotoxin could cause significant cell death and reduction of cell proliferation, as shown in the results determined by MTT assay, nitric oxide (NO) measurement and flow cytometric propidium iodine (PI) staining analysis, while pre‐treatment of PC12 cell with either BFP or its main ingredients prevented the toxicity to some degree. In addition, the neurotoxin caused an elevated activation of caspase‐3, the key enzyme for activation of the cellular apoptotic cascade, whereas BFP or its main ingredients inhibited the activation of caspase‐3. These results strongly indicate that BFP and its main ingredients may provide a useful therapeutic strategy for the treatment of neurodegenerative diseases, such as Parkinsons disease.

Differential effects of Matrigel and its components on functional activity of CFTR and ENaC in mouse endometrial epithelial cells.

Our previous studies have observed an effect of Matrigel, a solubilized basement membrane preparation extracted from the Engelbreth—Holm—Swarm (EHS) mouse sarcoma, on the expression of ion channels in mouse endometrial epithelia; namely the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP‐dependent Cl−channel, and the epithelial sodium channel (ENaC). The present study further investigated the effects of Matrigel and its individual components on the functional expression of CFTR and ENaC using the short‐circuit current (Isc) technique. The results showed that different components of Matrigel, namely growth factors, laminin and collagen, had differential effects on the functional activity of the two ion channels in murine endometrial epithelium. The information obtained may be useful for designing future in vitro culture models to investigate the functional roles of these ion channels in the endometrium.

Bak Foong Pills stimulate anion secretion across normal and cystic fibrosis pancreatic duct epithelia

The present study examined the effect of Bak Foong Pills (BFP), an over‐the‐counter traditional Chinese medicine (China registration no. Z980035), on anion secretion and the underlying signaling pathways in normal and cystic fibrosis pancreatic duct cell lines, CAPAN‐1 and CFPAC‐1, respectively, using the short‐circuit current technique. Apical addition of BFP ethanol extract (600μg/ml) induced a fast transient ISC peak that was followed by a slower but more sustained increase in ISC in CAPAN‐1 cells. However, the response to BFP in CFPAC‐1 was predominantly the first transient peak. Apical addition of DIDS (200μM) inhibited the first peak by more than 60% in both cell lines without significantly affecting the second ISC rise. More than 85% of the BFP‐induced first transient in both cell lines was inhibited when extra and intracellular Ca2+ was chelated or emptied by pre‐treatment with BAPTA (100μM) and thapsigargin (10μM), respectively. Acute addition of PMA (1μM), a PKC activator, blocked more than 95% of the BFP‐induced first peak in both cell lines, consistent with previously reported PKC modulation of Ca2+‐dependent pancreatic anion secretion. The BFP‐induced second ISC rise in CAPAN‐1 could be inhibited by 73.6% and 71.13% by pretreatment of the cells with MDL‐12330A (20μM), an adenylate cyclase inhibitor and Rp‐cAMP (200μM), a cyclic AMP antagonist, respectively. However, less than 25% of the ISC was inhibited by combined treatment with BAPTA and thapsigargin. The second rise was also completely blocked by DPC (2mM) or Glibenclamide (1mM). The results indicate that BFP ethanol extract stimulates pancreatic duct anion secretion in normal and CF cells via different signaling pathways involving both Ca2+ and cAMP.

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. Johns 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 GABAA receptor. This effect of myricetin was not blocked by the GABAA receptor benzodiazepine- (BZ-) binding site antagonist flumazenil, but by KN-62, a specific inhibitor of the Ca2+/calmodulin-stimulated protein kinase II (CaMK-II). Patch clamp and live Ca2+ imaging studies found that myricetin could increase Ca2+ current and intracellular Ca2+ concentration, respectively, via T- and L-type Ca2+ 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 GABAA receptor activity via calcium channel/CaMK-II dependent mechanism, which is distinctively different from that of most existing BZ-binding site agonists of GABAA receptor.

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 Parkinsons 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.

Traditional Chinese medicine Bak Foong Pills alters uterine quiescence – Possible role in alleviation of dysmenorrhoeal symptoms

Since contractility of the uterus appears to be the major source of pain during dysmenorrhoea, alleviation of the contractions is believed to be a possible treatment strategy. Bak Foong Pills, a traditional Chinese formulation for use in gynaecological disorders, has long been thought as effective in the treatment of dysmenorrhoeal symptoms. The present study thus aims to investigate whether ethanol extract of Bak Foong Pills (BFP‐Ex) or its constituent herbs may have direct effects on alleviating dysmenorrhoeal symptoms by altering uterine tone. This was investigated using isolated uterine preparations and intracellular messenger analysis of adenylate cyclase, via [3H]‐adenine assay, and calcium, with fluorometry imaging, in myometrial cultures. BFP‐Ex can stimulate uterine relaxation following oxytocin‐induced contractions ex‐vivo. Attempted inhibition of BFP‐Exs relaxatory response with a nitric oxide inhibitor and adenylate cyclase inhibitor, however, had no significant effect, suggesting that most of BFP‐Exs relaxatory response was not due to increases in NO or cAMP. Further studies on tetramethylpyrazine (TMP), a major active ingredient of BFP‐Ex, indicated that TMP could modulate intracellular calcium levels in favour of uteri relaxation. The ability of Bak Foong Pills to alleviate menstrual pain may be due to direct regulation of uterine tone.