Icariin inhibits the expression of IL-1ß, IL-6 and TNF-α induced by OGD/R through the IRE1/XBP1s pathway in microglia.

CONTEXT: Icariin (ICA), a flavonol glycoside extracted from Epimedium brevicornum Maxim (Berberidaceae), has been proven to inhibit inflammatory response in ischaemic rats in our laboratory's previous work. However, its underlying mechanism is still unclear. OBJECTIVE: This study investigates the effects of ICA on endoplasmic reticulum (ER) stress mediated inflammation induced by cerebral ischaemia-reperfusion (I/R) injury in vitro. MATERIALS AND METHODS: The primary cultured microglia were treated with oxygen-glucose deprivation (OGD) for 2 h followed by a 24 h reoxygenation. ICA (0.37, 0.74 and 1.48 µmol/L) administration was performed 1 h prior OGD and acting through 2 h OGD. The control group was cultured in normal conditions. At 24 h after reoxygenation, the expression of IRE1α, XBP1u, XBP1s, NLRP3 and caspase-1 was detected by western blotting (WB) and quantitative real-time (qRT) PCR; the expression of p-IRE1α was examined by WB; the expression of IL-1ß, IL-6 and TNF-α was measured by WB and enzyme-linked immunosorbent assay (ELISA). RESULTS: ICA (0.37, 0.74 and 1.48 µmol/L) reduced the ratio of p-IRE1α/IRE1α, the mRNA level of IRE1α, the expression of XBP1u, XBP1s, NLRP3, caspase-1 at both the mRNA and protein level expression of IL-1ß, IL-6 and TNF-α in OGD/R injured microglia. Overexpression of IRE1 significantly reversed the effects of ICA. DISCUSSION AND CONCLUSIONS: These results suggested that ICA might decrease the expression of IL-1ß, IL-6 and TNF-α by inhibiting IRE1/XBP1s pathway. The anti-inflammatory effect of ICA may provide a potential therapeutic strategy for the treatment of brain injury after stroke.

Icariin protects neurons from endoplasmic reticulum stress-induced apoptosis after OGD/R injury via suppressing IRE1α-XBP1 signaling pathway.

Icariin (ICA), a flavonol glycoside isolated from Epimedium, has been considered as a potential alternative therapy for ischemic stroke. However, the protective mechanisms of ICA on cerebral ischemia-reperfusion (I/R) are not fully illuminated yet. The effects of ICA on ER stress and inflammatory response which were involved in the pathological process of cerebral I/R were investigated in vitro. Microglia and neurons were subjected to OGD/R. ICA was administrated to microglia 1 h before OGD and maintained 2 h throughout OGD. At 24 h after reoxygenation, the protein expression of IL-1 ß, IL-6, TNF-α in the supernatant of microglia was measured using ELISA assay; neuronal apoptosis was assessed by TUNEL staining; and cell viability was detected using CKK-8 assay; the expression of IRE1α, XBP1u, XBP1s, and cleaved caspase-3 in neurons was examined by western blotting and qRT-PCR; the expression of p-IRE1α in neurons was detected by western blotting. We found that OGD/R induced the expression of IL-1 ß, IL-6, TNF-α in the supernatant of microglia; OGD/R and these proinflammatory cytokines promoted the mRNA as well as protein expression of XBP1u, XBP1s and cleaved caspase-3, increased the ratio of p-IRE1α/IRE1α, as well as apoptosis, and decreased cell viability in primary cortical neurons, while ICA reversed the levels of the above factors. IRE1 overexpression enhanced ER stress as well as apoptosis, and impaired the protective effects of ICA. These results suggested that ICA can inhibit apoptosis in neurons after OGD/R through IRE1/XBP1 signaling pathway beside its anti-inflammatory effect.

The effects of icariin on the expression of HIF-1α, HSP-60 and HSP-70 in PC12 cells suffered from oxygen-glucose deprivation-induced injury.

CONTEXT: The effects of icariin, a chief constituent of flavonoids from Epimedium brevicornum Maxim (Berberidaceae), on the levels of HIF-1α, HSP-60 and HSP-70 remain unknown. OBJECTIVE: To explore the effects of icariin on the levels of HSP-60, HIF-1α and HSP-70 neuron-specific enolase (NSE) and cell viability. MATERIALS AND METHODS: PC12 cells were treated with icariin (10-7, 10-6 or 10-5 mol/L) for 3 h (1 h before oxygen-glucose deprivation (OGD) plus 2 h OGD). HSP-60, HIF-1α, HSP-70 and NSE were measured using enzyme-linked immunosorbent assay (ELISA). Cell viability was determined by metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: After 2 h OGD, levels of HIF-1α, HSP-60, HSP-70 and NSE were increased significantly (HIF-1α: 33.3 ± 1.9 ng/L, HSP-60: 199 ± 16 ng/L, HSP-70: 195 ± 17 ng/L, NSE: 1487 ± 125 ng/L), and cell viability was significantly decreased (0.26 ± 0.03), while icariin (10-7, 10-6, or 10-5 mol/L) significantly reduced the contents of HIF-1α, HSP-60, HSP-70 and NSE (HIF-1α: 14.1 ± 1.4, 22.6 ± 1.8, 15.7 ± 2.1, HSP-60: 100 ± 12, 89 ± 6, 113 ± 11, HSP-70: 139 ± 9, 118 ± 7, 95 ± 9 and NSE: 1121 ± 80, 1019 ± 52, 731 ± 88), and improved cell viability (0.36 ± 0.03, 0.38 ± 0.04, 0.37 ± 0.03) in OGD-treated PC12 cells. DISCUSSION AND CONCLUSION: These results indicate that the protective mechanisms of icariin against OGD-induced injury may be related to down-regulating the expression of HIF-1α, HSP-60 and HSP-70.

Icariin Attenuates OGD/R-Induced Autophagy via Bcl-2-Dependent Cross Talk between Apoptosis and Autophagy in PC12 Cells.

Icariin (ICA), an active component of Epimedium brevicornum Maxim, exerts a variety of neuroprotective effects such as antiapoptosis. However, the mechanisms underlying antiapoptosis of ICA in neurons exposed to oxygen-glucose deprivation and reperfusion (OGD/R) are unclear. The B-cell lymphoma-2 (Bcl-2) protein family plays an important role in the regulation of apoptosis and autophagy through Bcl-2-dependent cross talk. Bcl-2 suppresses apoptosis by binding to Bax and inhibits autophagy by binding to Beclin-1 which is an autophagy related protein. In the present study, MTT result showed that ICA increased cell viability significantly in OGD/R treated PC12 cells (P < 0.01). Results of western blotting analysis showed that ICA increased Bcl-2 expression significantly and decreased expressions of Bax, cleaved Caspase-3, Beclin-1, and LC3-II significantly in OGD/R treated PC12 cells (P < 0.01). These results suggest that ICA protects PC12 cells from OGD/R induced autophagy via Bcl-2-dependent cross talk between apoptosis and autophagy.

ß-Asarone protects PC12 cells against OGD/R-induced injury via attenuating Beclin-1-dependent autophagy.

AIM: To explore the effects of ß-asarone from Acorus Tatarinowii Schott on autophagy in an ischemic stroke model of PC12 cells. METHODS: The ischemic stroke model of PC12 cells was made by OGD/R (2 h oxygen-glucose deprivation followed by 24 h reperfusion). Drug administration was started 1 h before OGD and last for 3 h. Then the cells were incubated in the drug-free and full culture medium under normoxic conditions for 24 h. After the treatments, Beclin-1, intracellular free calcium concentration ([Ca(2+)](i)) and mitochondrial membrane potential (MMP) were analyzed using flow cytometry. Cell viability was measured using MTT assay. Cell morphology was studied under inverted phase contrast microscope, and autophagosomes were observed under transmission electron microscope. RESULTS: Pretreatment with ß-asarone (20, 30, or 45 µg/mL) or the calcium channel antagonist nimodipine (10 µmol/L) significantly increased the cell viability and MMP, and decreased Beclin-1 expression and [Ca(2+)](i) in OGD/R-treated PC12 cells. Under inverted phase contrast microscope, pretreatment with ß-asarone or nimodipine dramatically increase the number of cells and improved the cellular morphology. Autophagosomes were found in OGD/R-treated PC12 cells as well as in drug plus OGD/R-treated PC12 cells. CONCLUSION: ß-Asarone protects PC12 cells against OGD/R-induced injury partly due to attenuating Beclin-1-dependent autophagy caused by decreasing [Ca(2+)](i) and increasing MMP.