Hydroxysafflor yellow A suppresses inflammatory responses of BV2 microglia after oxygen-glucose deprivation.

Inflammation is a pivotal pathological progress in the development of ischemic stroke. Modulating inflammatory cytokines released by microglia is thought to be a potential strategy for the treatment of ischemic stroke. Hydroxy-safflor yellow A (HSYA), a chemical component of the safflower yellow pigments, was reported to protect against brain injury in experimental stroke through anti-inflammation. However, the direct effect of HSYA on microglia following ischemia is unknown. This study confirmed whether HSYA could suppress inflammatory responses of BV2 microglia after oxygen glucose deprivation (OGD). BV2 microglia viability after OGD with or without HSYA was measured by MTT assay, PI/Annexin staining and LDH assay. Pro-inflammatory cytokines including 1L-1β, TNF-α, iNOS, COX-2, MCP-1 were determined by RT-PCR and western blotting. Activity of NF-κB and MAPK pathway were detected by western blotting. The results demonstrated that HSYA improved the viability of BV2 cells 12h after OGD with the profound dosage at 100mg/L by MTT assay. This observation was also confirmed by PI/Annexin staining and LDH assay. HSYA decreased the mRNA level of 1L-1β, TNF-α, iNOS, COX-2, MCP-1 and protein level of iNOS, COX-2 in BV2 microglia 12h after OGD. OGD enhanced the phosphorylation of p38 and nuclear translocation of p65 in BV2 microglia, which was partially reserved by HSYA. Our results suggested that HSYA suppressed inflammatory responses in BV2 microglia induced by OGD, which is probably associated with the inhibition of the NF-κB signaling pathway and phosphorylation of p38.

Li J ，Zhang S ，Lu M ，Chen Z ，Chen C ，Han L ，Zhang M ，Xu Y ... -  《-》

Hydroxy-safflor yellow A inhibits neuroinflammation mediated by Aβ₁₋₄₂ in BV-2 cells.

Inflammation is an important contributor to the development of Alzheimer's disease (AD). Anti-inflammatory medication may offer promising treatment for AD. Hydroxy-safflor yellow A (HSYA), a chemical component of the safflower yellow pigments, has been reported to exert potent immunosuppressive effects. This study examined the anti-inflammatory effects of HSYA in Aβ₁₋₄₂-treated BV-2 microglia cells. The mRNA levels of IL-1β, IL-4, IL-10, TNF-α, COX-2 and iNOS were detected by real-time PCR. Western blotting was used to determine the protein expression of COX-2, TNF-α, iNOS, Janus Kinase 2 (JAK2), p-JAK2, signal transducers and activators of transcription 3 (STAT3) and p-STAT3. BV2-conditioned medium was used to treat SH-SY5Y cells and primary neuronal cells in indirect toxicity experiments. Cell viability and apoptosis were assessed using MTT assay and Annexin V/PI staining respectively. The results demonstrated that HSYA significantly reduced the expression of the pro-inflammatory mediators and inhibited Aβ₁₋₄₂-induced neuroinflammation. Moreover, HSYA protected primary cortical neurons and SH-SY5Y cells against microglia-mediated neurotoxicity. HSYA also enhanced the phosphorylation of JAK2/STAT3 pathway and inhibition of JAK2 by AG 490 attenuated the anti-inflammatory effects of HSYA. Overall, our findings suggested that HSYA inhibited Aβ₁₋₄₂-induced inflammation and conferred neuroprotection partially through JAK2/STAT3 pathway, indicating that HSYA could be a potential drug for the treatment of AD.

Zhang Z ，Wu Z ，Zhu X ，Hui X ，Pan J ，Xu Y ... -  《-》

Hydroxy-safflor yellow A attenuates Aβ₁₋₄₂-induced inflammation by modulating the JAK2/STAT3/NF-κB pathway.

Beta-amyloid (Aβ)-mediated inflammation plays a critical role in the initiation and progression of Alzheimer׳s disease (AD). Anti-inflammatory treatment may provide therapeutic benefits. In this study, the effect of hydroxy-safflor yellow A (HSYA) on Aβ1-42-induced inflammation in AD mice was investigated and the underlying mechanisms were explored. Aβ1-42 was injected into bilateral hippocampi of mice to induce AD models in vivo. Spatial learning and memory of mice were investigated by the Morris water maze test. Activated microglia and astrocytes were examined by immunofluorescence staining for ionized calcium-binding adapter molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP). The mRNA of inflammatory cytokines were measured using real-time PCR. NF-κB p65 translocation was analyzed by western blotting and immunostaining. IκB and phosphorylation of JAK2 and STAT3 were tested by western blotting. The results showed that HSYA ameliorated the memory deficits in Aβ1-42-induced AD mice. HSYA suppressed Aβ1-42-induced activation of microglia and astrocytes and reduced the mRNA expression of pro-inflammatory mediators. HSYA up-regulated the JAK2/STAT3 pathway and inhibits the activation of NF-κB signaling pathways. Pharmacological inhibition of STAT3 by AG490 reversed the inactivation of p65 and anti-inflammatory effects of HSYA. In conclusion, these results suggest that HSYA protects Aβ1-42-induced AD model through inhibiting inflammatory response, which may involve the JAK2/STAT3/NF-κB pathway.

Zhang ZH ，Yu LJ ，Hui XC ，Wu ZZ ，Yin KL ，Yang H ，Xu Y ... -  《-》

The ability of hydroxysafflor yellow a to attenuate lipopolysaccharide-induced pulmonary inflammatory injury in mice.

Hydroxysafflor yellow A (HSYA) is a component of the flower of Carthamus tinctorius L. The present study investigated whether HSYA could attenuate acute lung injury (ALI) induced by lipopolysaccharide (LPS) administration. Male Kunming mice were pretreated with HSYA 0.5 h prior to intraperitoneal application of LPS. Arterial blood gas, lung water content index, lung tissue myeloperoxidase (MPO) activity, mRNA expression of inflammatory cytokines, NF-κBp65, p38 mitogen-activated protein kinase (MAPK) and pathological changes in lung morphology were assessed. After LPS administration, all animals displayed increased arterial carbon dioxide partial pressure (PaCO₂), and decreased arterial oxygen partial pressure (PaO₂), arterial oxygen saturation (SO₂), HCO₃⁻ concentration and pH, which were ameliorated by pretreating the animals with HSYA. HSYA administration significantly attenuated inflammatory cell infiltration and alleviated pulmonary edema induced by LPS. Moreover, HSYA decreased NF-κB p65 nuclear translocation, inhibited proinflammatory cytokine TNF-α, IL-1β and IL-6 mRNA expression and promoted antiinflammatory cytokine IL-10 gene expression following LPS injection. Pulmonary p38 MAPK phosphorylation was upregulated 4 h after LPS treatment, which could be suppressed by pretreatment with HSYA. These findings demonstrated the protective effect of HSYA against LPS-induced acute lung injury, which is suggested to be associated with the inhibition of p38 MAPK, NF-κB p65 activation and alteration of inflammatory cytokine expression.

Sun CY ，Pei CQ ，Zang BX ，Wang L ，Jin M ... -  《-》

Madecassoside protects BV2 microglial cells from oxygen-glucose deprivation/reperfusion-induced injury via inhibition of the toll-like receptor 4 signaling pathway.

In a previous study, the authors reported that madecassoside (MA) exerted a potent neuroprotective effect against cerebral ischemia-reperfusion (I/R) injury in rats, mediated by anti-oxidative, anti-inflammatory, and anti-apoptotic mechanisms. However, the cellular and molecular bases for its neuroprotective effects have not been fully elucidated. In this study, an in vitro ischemic model of oxygen-glucose deprivation followed by reperfusion (OGD/R) was used to investigate the role of the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB) pathway in the neuroprotective and anti-inflammatory effects of MA. BV2 microglia viability after OGD/R, treated with or without MA, was measured using the MTT assay. Messenger RNA and protein expression of pro-inflammatory cytokines (tumor necrosis factor α [TNF-α], interleukin-1β [IL-1β], interleukin-6 [IL-6]) were measured using real-time polymerase chain reaction (RT-PCR) and ELISA after OGD/R or lipopolysaccharide treatment. Expression of TLR4/MyD88 and NF-κB p65 were measured using RT-PCR, Western blotting, and immunofluorescence analysis. MA significantly rescued OGD/R-induced cytotoxicity in BV2 microglia. Meanwhile, MA suppressed the secretion of pro-inflammatory mediators, including TNF-α, IL-1β, and IL-6, induced by OGD/R or lipopolysaccharide in BV2 microglia. The mechanism of its neuroprotection and anti-inflammation from OGD/R may involve the inhibition of activation of TLR4 and MyD88 in BV2 microglia, and the blockage of NF-κB p65 nuclear translocation. MA exhibited a significant neuroprotective effect against I/R injury in both in vivo and in vitro experiments by attenuating microglia-mediated neuroinflammation via inhibition of the TLR4/MyD88/NF-κB signaling pathway.

Luo Y ，Wang C ，Li WH ，Liu J ，He HH ，Long JH ，Yang J ，Sui X ，Wang S ，You Z ，Wang YA ... -  《-》