Fingerprint analysis and multi-component determination of Zibu Piyin recipe by HPLC with DAD and Q-TOF/MS method

<p>Zibu Piyin recipe (ZBPYR), a traditional Chinese medicine formula, is used for curing dementia caused by diabetes. For quality control of ZBPYR, fingerprint analysis and qualitative analysis using high-performance liquid chromatography (HPLC) with a diode-array detector, and confirmation using HPLC coupled with electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS) were undertaken. HPLC fingerprint consisting of 34 common peaks was developed among 10 batches of ZBPYR, in which 7 common peaks were identified in comparison with the authentic standards and detected simultaneously. Furthermore, these seven compounds were verified by HPLC-Q-TOF-MS methods. The method can be applied to the quality control of ZBPYR.</p

Epiregulin (EPR) and amphiregulin (AR) inhibited cell death in Neuro2a cells.

<p>The cells were pretreated with EPR (0.01, 0.03, 0.1, and 1 nM) and AR (0.001, 0.01, 0.03, 0.1, and 1 nM) for 1 h and subsequently treated according to the indicated conditions. (A) EPR reduced the release of lactate dehydrogenase (LDH) at 48 h after Tm (1 μg/mL) treatment. (B) AR reduced the release of LDH at 48 h after Tm (1 μg/mL) treatment. The LDH released into the medium was expressed as a percentage of the control value. The data represent the average of 4 independent experiments for each sample. *p < 0.05.</p

Epiregulin (EPR) and amphiregulin (AR) inhibited endoplasmic reticulum stress in Neuro2a cells.

<p>Neuro2a cells were pre-incubated with (A) EPR or (B) AR for 1 h and then treated with tunicamycin (Tm) for 24 h. GRP78 and CHOP expression was detected via western blotting. Protein quantification is expressed as the mean ± standard error of 3 independent experiments. *p < 0.05 compared with the Tm-treated group.</p

Fold changes in EGF family member expression following LPS stimulation.

<p>Fold changes in EGF family member expression following LPS stimulation.</p

Stress-Induced Neuroprotective Effects of Epiregulin and Amphiregulin

<div><p>Members of the epidermal growth factor family play important roles in the regulation of cell growth, proliferation, and survival. However, the specific roles of each epidermal growth factor family member with respect to brain injury are not well understood. Gene chip assay screens have revealed drastic increases in the expression of the epidermal growth factor family members amphiregulin and epiregulin following lipopolysaccharide stimulation, which activates an immune response. Both immune activity and endoplasmic reticulum stress are activated during cerebral ischemia. We found that the expression levels of amphiregulin and epiregulin were significantly increased under conditions of cerebral ischemia. Because endoplasmic reticulum stress increased the expression of amphiregulin and epiregulin in glial cells, endoplasmic reticulum stress may be a key mediatory factor of pathophysiological activity. Recombinant epiregulin and amphiregulin proteins effectively inhibited endoplasmic reticulum stress and the subsequent induction of neuronal cell death. Therefore, the upregulation of the epidermal growth factor family members epiregulin and amphiregulin may play a critical role in preventing endoplasmic reticulum stress-induced cell death, thus providing a potential therapy for brain injury.</p></div

Induction of epiregulin (EPR) and amphiregulin (AR) mRNA expression in primary glial cells following Tm treatment.

<p>Total RNA was isolated from cells exposed to Tm (3 μg/ml) for the indicated periods and subjected to RT-PCR. Data are presented as the means ± standard errors from 3 separate experiments. *p < 0.05 compared with the control.</p

Induction of epiregulin (EPR) and amphiregulin (AR) mRNA expression in primary glial cells under hypoxic conditions.

<p>Total RNA was isolated from cells exposed to hypoxic conditions for the indicated time periods and subjected to RT-PCR. Data are presented as the means ± standard errors from 3 separate experiments. *p < 0.05 compared with the control.</p

Epiregulin (EPR) and amphiregulin (AR) inhibited cell death in Neuro2a cells.

<p>The cells were pretreated with EPR (0.01, 0.03, 0.1, and 1 nM) and AR (0.001, 0.01, 0.03, 0.1, and 1 nM) for 1 h and subsequently treated according to the indicated conditions. (A) EPR reduced the release of lactate dehydrogenase (LDH) at 48 h after Tm (1 μg/mL) treatment. (B) AR reduced the release of LDH at 48 h after Tm (1 μg/mL) treatment. The LDH released into the medium was expressed as a percentage of the control value. The data represent the average of 4 independent experiments for each sample. *p < 0.05.</p

Induction of epiregulin (EPR) and amphiregulin (AR) mRNA expression in primary glial cells following lipopolysaccharide (LPS) treatment (time course and dose course).

<p>(A) Total RNA was isolated from cells exposed to LPS (1 μg/ml) for the indicated time periods and subjected to RT-PCR. (B) Total RNA was isolated from cells exposed to LPS (4 h) at the indicated concentrations and subjected to RT-PCR. Data are presented as the means ± standard errors from 3 separate experiments. *p < 0.05 compared with the control.</p

Fold changes in pro-inflammatory cytokines expression following LPS stimulation.

<p>Fold changes in pro-inflammatory cytokines expression following LPS stimulation.</p