Stroke is a serious disease with complex pathomechanism and limited therapeutic effect in clinic. Our previous research has found obvious therapeutic effect of Puerarin (Pur) on stroke injury of rat. The aim of this study is to investigate the transcriptome changes of oxygen-glucose deprivation/reoxygenation (OGD/R)-injured astrocytes before and after the intervention of Pur. Cells activity and apoptosis detection indicated that the activity of OGD/R-injured astrocytes was improved, and the apoptosis was ameliorated by Pur. Affymetrix GeneChip Rat Genome 230 2.0 Array assays indicated that after intervention of Pur, mRNA expressions of 31 genes were up-regulated and 40 genes were down-regulated in OGD group, whereas mRNA expression of 36 genes were up-regulated, and 88 genes were down-regulated in OGD/R group. Pathway analysis indicated that the olfactory transduction pathway and the JAK (janus kinase) 2/STAT (signal transducer and activator of transcription) three pathways were down-regulated by Pur during OGD/R injury of astrocytes. These data indicated that Pur regulates transcriptome and expresses protective effect on astrocytes during OGD/R injury, and may be a potential therapeutic agent for the treatment of stroke.

This present paper focus on transcriptome changes of astrocytes before and after oxygen glucose deprivation (OGD) injury. Cortical astrocytes of Sprague-Dawley (SD) rats were cultured in OGD condition for 6 h, and the damage of cells was analyzed by cell’s activity detection, lactate dehydrogenase leakage rate detection, annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) apoptosis detection, and transcriptome change detection through Affymetrix GeneChip Rat Genome 230 2.0 Array; some regulated genes were detected by quantitative real-time polymerase chain reaction (qPCR). After OGD injury, activity of astrocytes was decreased, and apoptosis of injured cells was increased. mRNA expressions of 404 genes were upregulated and 454 genes were downregulated, including upregulation of MAPK signal pathway and downregulation of PI3k/Akt pathway. Otherwise, pathways of the focal adhesion (percentage of all regulated genes, 11.4 %), HIF-1 (10.2 %), and one carbon pool by folate (3.4 %), etc. were upregulated, and steroid biosynthesis (7 %), basal cell carcinoma (9.9 %), and terpenoid backbone (5.6 %), etc. were down-regulated. These results indicate that apoptosis of astrocytes induced by OGD injury is associated with the MAPK and PI3k/Akt pathways.

•Brain EPO expression was decreased in d-gal-induced aging model.•The decline of EPO was correlated with oxidative stress.•HIF-2a was involved in Oxidative stress inducing the decline of EPO.Brain Erythropoietin (EPO), an important neurotrophic factor and neuroprotective factor, was found to be associated with aging. Studies found EPO expression was significantly decreased in the hippocampus of aging rat compared with that of the youth. But mechanisms of the decline of the brain EPO during aging remain unclear. The present study utilized a d-galactose (d-gal)-induced aging model in which the inducement of aging was mainly oxidative injury, to explore underlying mechanisms for the decline of brain EPO in aging rats. d-gal-induced aging rats (2 months) were simulated by subcutaneously injecting with d-gal at doses of 50 mg·kg− 1, 150 mg·kg− 1 and 250 mg·kg− 1 daily for 8 weeks while the control group received vehicle only. These groups were all compared with the aging rats (24 months) which had received no other treatment. The cognitive impairment was assessed using Morris water maze (MWM) in the prepared models, and the amount of β-galactosidase, the lipid peroxidation product malondialdehyde (MDA) level and the superoxide dismutase (SOD) activity in the hippocampus was examined by assay kits. The levels of EPO, EPOR, p-JAK2 and hypoxia-inducible factor-2α (HIF-2α) in the hippocampus were detected by western blot. Additionally, the correlation coefficient between EPO/EPOR expression and MDA level was analyzed. The MWM test showed that compared to control group, the escape latency was significantly extended and the times of crossing the platform was decreased at the doses of 150 mg·kg− 1 and 250 mg·kg− 1 (p < 0.05). Also, the amount of β-galactosidase and the MDA level in the hippocampus were significantly increased but the SOD activity was significantly decreased (p < 0.05, 0.01 and 0.01, respectively). Similar to aging rats, the expressions of EPO, EPOR, p-JAK2, and HIF-2αin the brain of d-gal-treated rats were significantly decreased (p < 0.05) at 150 mg·kg− 1 and 250 mg·kg− 1. Interestingly, negative correlations were found between EPOR (r = − 0.699, p < 0.01), EPO (r = − 0.701, p < 0.01) and the MDA level. These results indicated that aging could result in the decline of EPO in the hippocampus and oxidative stress might be the main reason for the decline of brain EPO in aging rats, involved with the decrease of HIF-2α stability.

•Brain EPO expression was decreased in d-gal-induced aging model.•The decline of EPO was correlated with oxidative stress.•HIF-2a was involved in Oxidative stress inducing the decline of EPO.Brain Erythropoietin (EPO), an important neurotrophic factor and neuroprotective factor, was found to be associated with aging. Studies found EPO expression was significantly decreased in the hippocampus of aging rat compared with that of the youth. But mechanisms of the decline of the brain EPO during aging remain unclear. The present study utilized a d-galactose (d-gal)-induced aging model in which the inducement of aging was mainly oxidative injury, to explore underlying mechanisms for the decline of brain EPO in aging rats. d-gal-induced aging rats (2 months) were simulated by subcutaneously injecting with d-gal at doses of 50 mg·kg− 1, 150 mg·kg− 1 and 250 mg·kg− 1 daily for 8 weeks while the control group received vehicle only. These groups were all compared with the aging rats (24 months) which had received no other treatment. The cognitive impairment was assessed using Morris water maze (MWM) in the prepared models, and the amount of β-galactosidase, the lipid peroxidation product malondialdehyde (MDA) level and the superoxide dismutase (SOD) activity in the hippocampus was examined by assay kits. The levels of EPO, EPOR, p-JAK2 and hypoxia-inducible factor-2α (HIF-2α) in the hippocampus were detected by western blot. Additionally, the correlation coefficient between EPO/EPOR expression and MDA level was analyzed. The MWM test showed that compared to control group, the escape latency was significantly extended and the times of crossing the platform was decreased at the doses of 150 mg·kg− 1 and 250 mg·kg− 1 (p < 0.05). Also, the amount of β-galactosidase and the MDA level in the hippocampus were significantly increased but the SOD activity was significantly decreased (p < 0.05, 0.01 and 0.01, respectively). Similar to aging rats, the expressions of EPO, EPOR, p-JAK2, and HIF-2αin the brain of d-gal-treated rats were significantly decreased (p < 0.05) at 150 mg·kg− 1 and 250 mg·kg− 1. Interestingly, negative correlations were found between EPOR (r = − 0.699, p < 0.01), EPO (r = − 0.701, p < 0.01) and the MDA level. These results indicated that aging could result in the decline of EPO in the hippocampus and oxidative stress might be the main reason for the decline of brain EPO in aging rats, involved with the decrease of HIF-2α stability.

Ethnopharmacological relevanceDu Liang soft capsule (DL) is a traditional Chinese medicine for treating migraines; it is made from two Chinese herbs, including Ligusticum striatum DC., root; Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav., root.Aim of the studyIn the present study, we aimed to elucidate the pharmacodynamic action of DL and its mechanism in an animal model of migraines induced by glyceryl trinitrate (GTN).Materials and methodsSixty rats were randomly divided into six groups, including a normal control group, model control group, positive group (Sumatriptan 0.006 g kg−1), and three DL groups (0.44, 1.31 and 3.93 g kg−1). All rats were intragastrically treated with the corresponding treatment for 7 consecutive days, and they were subcutaneously injected with GTN (10 mg kg−1) 30 min after the last treatment, except in the normal control group. After model establishment, the behaviors of all rats, including head scratching, cage climbing, and the development of red ears were observed continuously by digital camera every 30 min for 3 h. Four hours after GTN treatment, all rats were anaesthetized and the blood and tissue samples were collected. Plasma calcitonin gene related to peptide (CGRP) and endothelin (ET) levels were measured using the radioimmunoassay method, and serum NO was determined by the colorimetric method. Afterwards, the brainstem tissues were dissected and washed with physiological saline, and divided evenly into two parts. One part was used to test the monoamine levels, including levels of 5-hydroxytryptamine (5-HT), norepinephrine (NE) and dopamine (DA), by the fluorometric method, and the other part was used to determine the nuclear factor kappaB (NF-κB) p65, nuclear c-fos, inducible nitric oxide synthase (iNOS), interleukin (IL)-1β (IL-1β), and cyclooxygenase-2 (COX-2) levels by Western blot analysis.ResultsIn the pharmacodynamic action assay, DL (1.31 and 3.93 g kg−1) greatly improved the abnormal behaviors of migraine rats, including head scratching and cage climbing, and the development of red ears. In the mechanism assay, compared with the control group, the plasma CGRP and serum NO levels and the brainstem 5-HT, NE and DA levels in the DL administration groups were significantly decreased; and the plasma ET levels were remarkably increased. Moreover, down-regulation of NF-κB p65, c-fos and pro-inflammatory cytokines, including iNOS, IL-1β and COX-2 in the brainstem in the DL administration groups were observed by Western blot analysis.ConclusionsThe above results suggested that DL has a therapeutic effect on migraines, and its mechanism may be related to adjusting the level of neurotransmitters and vasoactive substances, consequently relieving neurogenic inflammation.Download high-res image (276KB)Download full-size image

Ethnopharmacological relevanceFerulic acid (FA), ligustrazine (LZ) and tetrahydropalmatine (THP) are separately isolated from Chinese Angelica, Szechwan Lovage Rhizome and Rhizoma in the Jiawei-Foshou-San formula, a popular traditional Chinese medicine for irregular menses. It has been reported that the combination use of FA+LZ+THP has similar effect on endometriosis, but the underlying mechanisms are unclear. This study was to investigate the combination effects and mechanisms of FA+LZ+THP on endometriosis rats.Materials and methodsFifty endometriosis rats were intragastricly treated with FA+LZ+THP for 4 wk. The volume of ectopic endometrial tissue was measured to evaluate the effects. Then the changes in hypothalamic–pituitary–ovarian axis and ERE pathway were indicated by the levels of E2, GnRH, FSH and LH, and the expressions of ER, HSP90 and COX-2, respectively. In addition, peritoneal macrophages of each rat were cultured in vitro and treated with (FA+LZ+THP)-medicated serum for 24 h. The proliferation and phagocytosis abilities, the levels of IL-1β and TNF-α, and the expression of IκBα were then measured for the changes of peritoneal macrophage activities.ResultsCombination use of FA+LZ+THP diminished the volume of the ectopic endometrial tissues (P<0.05 or P<0.01). It also decreased the E2 level, suppressed the expression of GnRH, FSH and LH, and decreased the protein expression of ER, HSP90 and COX-2 (all P<0.05 or P<0.01). The phagocytosis ability of peritoneal macrophage was enhanced by (FA+LZ+THP)-medicated serum (P<0.05) with no change of proliferation (P>0.05). Moreover, IL-1β and TNF-α were downregulated (both P<0.05 or P<0.01) and IκBα was upregulated by the (FA+LZ+THP)-medicated serum (P<0.01).ConclusionsThe combination use of FA, LZ and THP could inhibit the growth of ectopic endometrial tissue in endometriosis rats. It might be related to the down-regulation of hypothalamic–pituitary–ovarian axis, the amelioration in ERE pathway and the improvement of peritoneal macrophage activities.Download high-res image (127KB)Download full-size image