PMExposure Elicits Oxidative Stress Responses and Mitochondrial Apoptosis Pathway Activation in HaCaT Keratinocytes / 中华医学杂志(英文版)

<p><b>Background:</b>PM(aerodynamic diameter ≤ 2.5 μm) is a dominant and ubiquitous air pollutant that has become a global concern as PMexposure has been linked to many adverse health effects including cardiovascular and pulmonary diseases. Emerging evidence supports a correlation between increased air PMlevels and skin disorders although reports on the underlying pathophysiological mechanisms are limited. Oxidative stress is the most common mechanism of PM-induced adverse health effects. This study aimed to investigate PM-induced oxidative damage and apoptosis in immortalized human keratinocyte (HaCaT) cells.</p><p><b>Methods:</b>HaCaT cells were exposed to 0, 25, 50, 100, or 200 μg/ml PMfor 24 h. Reactive oxygen species (ROS) generation, lipid peroxidation products, antioxidant activity, DNA damage, apoptotic protein expression, and cell apoptosis were measured.</p><p><b>Results:</b>PMexposure (0-200 μg/ml) for 24 h resulted in increased ROS levels (arbitrary unit: 201.00 ± 19.28, 264.50 ± 17.91, 305.05 ± 19.57, 427.95 ± 18.32, and 436.70 ± 17.77) and malondialdehyde production (0.54 ± 0.05 nmol/mg prot, 0.61 ± 0.06 nmol/mg prot, 0.68 ± 0.05 nmol/mg prot, 0.70 ± 0.05 nmol/mg prot, and 0.76 ± 0.05 nmol/mg prot), diminished superoxide dismutase activity (6.47 ± 0.28 NU/mg prot, 5.97 ± 0.30 NU/mg prot, 5.15 ± 0.42 NU/mg prot, 4.08 ± 0.20 NU/mg prot, and 3.76 ± 0.37 NU/mg prot), and increased DNA damage and apoptosis in a dose-dependent manner in HaCaT cells. Moreover, cytochrome-c, caspase-3, and caspase-9 expression also increased proportionately with PMdosing.</p><p><b>Conclusion:</b>PMmight elicit oxidative stress and mitochondria-dependent apoptosis that likely manifests as skin irritation and damage.</p>

Simultaneous determination of six components in Hedyotis diffusa Willd by HPLC / 中国药学杂志

OBJECTIVE: To develop an HPLC method for simultaneous determination of multiple-components in Hedyotis diffusa Willd. METHODS: The HPLC analysis was carried out on a C18 column (4.6 mm×250 mm, 5 μm) by gradient elution with acetoni-trile-water[both containing 0.1‰ (V/V) acetic acid] as mobile phase at a flow rate of 0.8 mL·min-1, the column temperature at 35°C, and the detection wavelength was set at 238 nm. External standard method and quantitative analysis of multi-components by single marker (QAMS) method were adopted for simultaneous determination of six components in Hedyotis diffusa Willd, respectively. RESULTS: The linear ranges for asperulosidic acid, quercetin-3-O-[2-O-(6-O-E-feruloyl)-β -D-glucopyranosyl]-β-D-glucopyrano-side, kaempferol-3-O-[2-O-(6-O-E-feruloyl)-β-Z) -gfucopyranosyl]-β-D-galactopyranoside, (E)-6-O-p-coumaroyl scandoside methyl ester, (E)-6-O-feruloyl scandoside methyl ester, (Z)-6-O-p-coumaroyl scandoside methyl ester were 2.34-93.50, 2.61-104.33, 0.67-26.69, 3.42-136.84, 0.65-26.07, and 1.10-44.17 μg·mL-1 (r<0.9993), respectively. The RSD values of precision, reproducibility, and sample stability were not more than 2.2%. The average recoveries of the six components were 99.8%-101.1% with RSDs not more than 1.2%. The P values of external standard method and QAMS by paired t-test were greater than 0.05. CONCLUSION: There is no significant difference in the content analysis results of the two methods, which can both used for simultaneous determination of the four iridoids and two flavonoids in Hedyotis diffusa Willd.

Alterations in gene expression and steroidogenesis in the testes of transient cerebral ischemia in male rats.

BACKGROUND: Serum testosterone levels have been found lower in acute ischemic stroke male patients. However, the exact mechanism remains unclear. In the present study, we measured serum testosterone levels, steroidogenesis- related genes and Leydig cells number in experimental transient cerebral ischemia male rats to elucidate the mechanism. METHODS: The middle cerebral arteries of adult male Sprague-Dawley rats were sutured for 120 minutes and then sacrificed after 24 hours. Blood was collected for measurement of serum testosterone, follicular stimulating hormone and estradiol levels, and testes were collected for measurement of steroidogenesis-related gene mRNA levels and number of Leydig cells. RESULTS: Serum testosterone levels in rats after cerebral ischemia were significantly lower (0.53 ± 0.16) ng/ml, n = 7, mean ± SE) compared with control ((2.33 ± 0.60) ng/ml, n = 7), while serum estradiol and follicular stimulating hormone levels did not change. The mRNA levels for luteinizing hormone receptor (Lhcgr), scavenger receptor class B member 1 (Scarb1), steroidogenic acute regulatory protein (StAR), cholesterol side chain cleavage enzyme (Cyp11a1), 3ß-hydroxysteroid dehydrogenase 1 (HSD3ß1), 17α-hydroxylase/20-lyase (Cyp17a1) and membrane receptor c-kit (kit) were significantly downregulated by cerebral ischemia, while luteinizing hormone, Kit ligand (KitL), 17ß-hydrosteroid dehydrogenase 3 (HSD17ß3) and 5α-reductase (Srd5a1) were not affected. We also observed that, relative to control, the Leydig cell number did not change. CONCLUSIONS: These results indicate that transient cerebral ischemia in the brain results in lower expression levels of steroidogenesis-related genes and thus lower serum testosterone level. Transient cerebral ischemia did not lower the number of Leydig cells.

Gephyrin interacts with the glutamate receptor interacting protein 1 isoforms at GABAergic synapses.

We have previously shown that the glutamate receptor interacting protein 1 (GRIP1) splice forms GRIP1a/b and GRIP1c4-7 are present at the GABAergic post-synaptic complex. Nevertheless, the role that these GRIP1 protein isoforms play at the GABAergic post-synaptic complex is not known. We are now showing that GRIP1c4-7 and GRIP1a/b interact with gephyrin, the main post-synaptic scaffold protein of GABAergic and glycinergic synapses. Gephyrin coprecipitates with GRIP1c4-7 or GRIP1a/b from rat brain extracts and from extracts of human embryonic kidney 293 cells that have been cotransfected with gephyrin and one of the GRIP1 protein isoforms. Moreover, purified gephyrin binds to purified GRIP1c4-7 or GRIP1a/b, indicating that gephyrin directly interacts with the common region of these GRIP1 proteins, which includes PDZ domains 4-7. An engineered deletion construct of GRIP1a/b (GRIP1a4-7), which both contains the aforementioned common region and binds to gephyrin, targets to the post-synaptic GABAergic complex of transfected cultured hippocampal neurons. In these hippocampal cultures, endogenous gephyrin colocalizes with endogenous GRIP1c4-7 and GRIP1a/b in over 90% of the GABAergic synapses. Double-labeling electron microscopy immunogold reveals that in the rat brain GRIP1c4-7 and GRIP1a/b colocalize with gephyrin at the post-synaptic complex of individual synapses. These results indicate that GRIP1c4-7 and GRIP1a/b colocalize and interact with gephyrin at the GABAergic post-synaptic complex and suggest that this interaction plays a role in GABAergic synaptic function.

Gephyrin clustering is required for the stability of GABAergic synapses.

Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. We report here that knocking down gephyrin expression with small hairpin RNAs (shRNAs) in cultured hippocampal pyramidal cells decreased both the number of gephyrin and GABA(A) receptor clusters. Similar results were obtained by disrupting the clustering of endogenous gephyrin by overexpressing a gephyrin-EGFP fusion protein that formed aggregates with the endogenous gephyrin. Disrupting postsynaptic gephyrin clusters also had transsynaptic effects leading to a significant reduction of GABAergic presynaptic boutons contacting the transfected pyramidal cells. Consistent with the morphological decrease of GABAergic synapses, electrophysiological analysis revealed a significant reduction in both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). However, no change in the whole-cell GABA currents was detected, suggesting a selective effect of gephyrin on GABA(A) receptor clustering at postsynaptic sites. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses.

Disruption of postsynaptic GABA receptor clusters leads to decreased GABAergic innervation of pyramidal neurons.

We have used RNA interference (RNAi) to knock down the expression of the gamma2 subunit of the GABA(A) receptors (GABA(A)Rs) in pyramidal neurons in culture and in the intact brain. Two hairpin small interference RNAs (shRNAs) for the gamma2 subunit, one targeting the coding region and the other one the 3'-untranslated region (UTR) of the gamma2 mRNA, when introduced into cultured rat hippocampal pyramidal neurons, efficiently inhibited the synthesis of the GABA(A) receptor gamma2 subunit and the clustering of other GABA(A)R subunits and gephyrin in these cells. More significantly, this effect was accompanied by a reduction of the GABAergic innervation that these neurons received. In contrast, the gamma2 shRNAs had no effect on the clustering of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, postsynaptic density protein 95 (PSD-95) or presynaptic glutamatergic innervation. A gamma2-enhanced green fluorescent protein (EGFP) subunit construct, whose mRNA did not contain the 3'-UTR targeted by gamma2 RNAi, rescued both the postsynaptic clustering of GABA(A)Rs and the GABAergic innervation. Decreased GABA(A)R clustering and GABAergic innervation of pyramidal neurons in the post-natal rat cerebral cortex was also observed after in utero transfection of these neurons with the gamma2 shRNAs. The results indicate that the postsynaptic clustering of GABA(A)Rs in pyramidal neurons is involved in the stabilization of the presynaptic GABAergic contacts.

GRIP1 in GABAergic synapses.

The glutamate receptor-interacting protein GRIP1 is present in glutamatergic synapses and interacts with the GluR2/3/4c subunits of the AMPA receptors. This interaction plays important roles in trafficking, synaptic targeting, and recycling of AMPA receptors as well as in the plasticity of glutamatergic synapses. Although GRIP1 has been shown to be present at GABAergic synapses in cultured neurons, the use of EM (electron microscopy) immunocytochemistry in the intact brain has failed to convincingly reveal the presence of GRIP1 in GABAergic synapses. Therefore, most studies on GRIP1 have focused on glutamatergic synapses. By using mild tissue fixation and embedding in EM, we show that in the intact brain the 7-PDZ domain GRIP1a/b is present not only in glutamatergic synapses but also in GABAergic synapses. In GABAergic synapses GRIP1a/b localizes both at the presynaptic terminals and postsynaptically, being frequently localized on the synaptic membranes or the synaptic junctional complex. Considerably higher density of GRIP1a/b is found in the presynaptic GABAergic terminals than in the glutamatergic terminals, while the density of GRIP1a/b in the postsynaptic complex is similar in both types of synapses. The results also show that the 7-PDZ and the shorter 4-PDZ domain splice forms of GRIP1 (GRIP1c 4-7) frequently colocalize with each other in individual GABAergic and glutamatergic synapses. The results suggest that GRIP1 splice forms might play important roles in brain GABAergic synapses.