Qingda granule exerts neuroprotective effects against ischemia/reperfusion-induced cerebral injury via lncRNA GAS5/miR-137 signaling pathway

Abstract

Background: Ischemic stroke is the second leading cause of death and disability worldwide, which needs to develop new pharmaceuticals for its prevention and treatment. Qingda granule (QDG), a traditional Chinese medicine formulation, could improve angiotensin II-induced brain injury and decrease systemic inflammation. In this study, we aimed to evaluate the neuroprotective effect of QDG against ischemia/reperfusion-induced cerebral injury and illustrate the potential mechanisms. Methods: The middle cerebral artery occlusion/reperfusion (MCAO/R) surgery in vivo and oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro models were established. Ischemic infarct volume was quantified using magnetic resonance imaging (MRI). Neurobehavioral deficits were assessed using a five-point scale. Cerebral histopathology was determined by hematoxylin-eosin (HE) staining. Neuronal apoptosis was evaluated by TUNEL and immunostaining with NeuN antibodies. The protective effect of QDG on OGD/R-injured HT22 cells was determined by MTT assay and Hoechst 33258 staining. The expression of lncRNA GAS5, miR-137 and apoptosis-related proteins were investigated in MCAO/R-injured rats and in OGD/R-injured HT22 cells using RT-qPCR and western blot analysis. Results: QDG significantly reduced the ischemic infarct volume, which was accompanied with improvements in neurobehavioral deficits. Additionally, QDG significantly ameliorated cerebral histopathological changes and reduced neuron loss in MCAO/R-injured rats. Moreover, QDG improved growth and inhibited apoptosis of HT22 cells injured by OGD/R in vitro. Finally, QDG significantly decreased the expression of lncRNA GAS5, Bax and cleaved caspase3, whereas it increased miR-137 and Bcl-2 expression in MCAO/R-injured rats and in OGD/R-injured HT22 cells. Conclusion: QDG plays a neuroprotective role in ischemic stroke via regulation of the lncRNA GAS5/miR-137 signaling pathway.

Keywords: Qingda granule; ischemic stroke; lncRNA GAS5; miR-137; neuronal apoptosis.

Figure 1

QDG decreases OGD/R-induced cell damage in HT22 cells. (A) Cytotoxicity of QDG on HT22 cells. (B) Effects of QDG on OGD/R-induced cell damage in HT22 cells. The cell viability was measured by MTT assay. Data are presented as means ± standard deviations. *p<0.05, compared to control group; Δp<0.05, compared to OGD/R group.

Figure 2

QDG attenuates weight loss and neurological deficits in rats after MCAO/R surgery. (A) The weight loss of rats in each group was measured daily. (B) The neurobehavioral score of rats in each group was assessed daily after the MCAO/R surgery using a five-point scale. *p<0.05, compared to sham group; △p<0.05, compared to model group.

Figure 3

QDG reduces infarct volume and improves cerebral histopathology in rats after MCAO/R surgery. (A and B) Representative images of brain infarct volume were determined by MRI scanning at the 1^st, 3^rd, and 7^th day after MCAO/R surgery and the percentage of infarct volume was analyzed via Image J software. Data are presented as means ± standard deviations of eight rats in each group. *p<0.05 compared to sham group; Δp<0.05, compared to model group. (C) Representative images are shown and were taken at a magnification of 40× by HE staining.

Figure 4

QDG attenuates neuronal apoptosis and loss in rats after MCAO/R surgery and in HT22 cells after OGD/R. (A) Representative images of apoptosis in the cerebral cortex area are presented at a magnification of 40× using TUNEL staining. (B) Representative images of NeuN-positive neurons in the cerebral cortex area are presented at a magnification of 40× using IHC analysis. (C) Representative images of HT22 cells apoptosis are presented at a magnification of 20× using Hoechst 33258 staining. (D) The corresponding quantification of apoptosis in the cerebral cortex area was determined by TUNEL staining. (E) The corresponding quantification of NeuN immunoreactivity was determined by IHC analysis. (F) The corresponding quantification of HT22 cells apoptosis was determined by Hoechst 33258 staining. *p<0.05 compared to sham group; Δp<0.05, compared to model group; *p<0.05, compared to control group; Δp<0.05, compared to OGD/R group.

Figure 5

QDG modulates apoptotic-regulatory proteins in MCAO/R-injured rats and in OGD/R-injured HT22 cells. (A and B) Protein expression of Bax, Bcl-2, and cleaved caspase3 were determined by western blot analysis. The internal control used was β-actin. (C and D) Relative densitometric findings of the above proteins were determined via Image Lab software. *p<0.05, compared to sham group; △p<0.05, compared to model group; *p<0.05, compared to control group; △p<0.05, compared to OGD/R group.

Figure 6

QDG targets the lncRNA GAS5/miR-137 axis in MCAO/R-injured rats and in OGD/R- injured HT22 cells. (A and B) The expression of lncRNA GAS5 was determined using RT-qPCR analysis with GAPDH as the internal control. (C and D) The expression of miR-137 was determined using RT-qPCR analysis with U6 as an internal control. *p<0.05 compared to sham group; △p<0.05, compared to model group; *p<0.05, compared to control group; △p<0.05, compared to OGD/R group.