The present study showed that OXA protected the brain against I/R injury by reducing cerebral infarction volume and improving neurological function score, which is consistent with previous reports [27, 28]. However, the highlight is that we found that OXA exerted anti-inflammatory effects by reducing the expression of inflammatory factors, and inhibited the excessive activation of astrocytes in the cortex of rats after cerebral I/R injury. As an important neuroprotective agent, OXA has a significant effect of anti-apoptosis consistent with our prior research . Here, we found that OXA could effectively reduce apoptosis expression and inflammation caused by OGD/R in U251 astrocytoma cells. To the best of our knowledge, this is the first research focusing on the mechanisms of the effects of OXA on astrocytes.
Increasing evidences have suggests that the inflammatory response leads to secondary brain damage following cerebral I/R . Immune effector cells from the cerebral cortex will produce and release a large number of pro-inflammatory factors, such as IL-1β and TNF-α. These pro-inflammatory factors will initiate and aggravate the inflammatory response and further aggravate brain damage . Therefore, reducing the production and release of pro-inflammatory factors is an effective means to alleviate cerebral I/R injury. Inflammation is also considered to be one of the main targets for the development of new stroke treatments . Previous reports have shown that treatment with exogenous IL-1β can aggravate brain damage . TNF-α is one of the most widely studied cytokines. Administration of TNF-α during ischemic brain injury could exacerbate the damage, and administration of TNF-neutralizing antibodies can reduce this damage . The up-regulation of iNOS expression can lead to excessive production of NO after cerebral ischemia, and the massive production of NO causes irreversible damage to cells by inhibiting the mitochondrial respiratory chain and forming peroxynitrite with superoxide anions [42–44]. Moreover, a growing amount of evidence demonstrates that neurotoxicity caused by ischemia could be alleviated by iNOS inhibitors [45, 46]. In this study we found that OXA treatment significantly decreased expression levels of TNF-α, IL-1β, IL-6 and iNOS induced by MCAO/R. These results show that OXA has neuroprotective effects against I/R injury by preventing pro-inflammatory cytokines.
Astrocytes are the most ubiquitous type of nerve cells in the brain, which are implicated in pathological process of ischemic stroke. Cerebral ischemia activates astrocytes. However, reactive astrocytes are considered to be detrimental to the neurological outcome after stroke . The activated astrocytes release many pro-inflammatory factors, and then a large number of pro-inflammatory factors trigger and amplify the inflammatory response, and further activate astrocytes to form a cycle . It is generally believed that reactive astrocytes in the early stage of central nervous system damage are beneficial to neurons. They support neurons by participating in a variety of biological processes such as regulating inflammation and neurotransmitter nutrition. However, over-activated astrocytes can accelerate the process of neuronal death and infarction [9, 47]. Astrocyte activation is manifested by astrocyte proliferation, morphological changes and enhanced expression of GFAP. In this study, we observed the proliferation of astrocytes in the infarcted cortex, and the expression of astrocyte marker GFAP also increased significantly, which was abolished by the OXA treatment. This indicates that the activation of astrocytes plays an important role in cerebral I/R injury, and OXA inhibits the excessive activation of astrocytes to exert neuroprotective effects. Accumulating evidence strongly suggests that astrocyte-mediated inflammation can cause secondary damage to the brain after stroke [12, 13]. Therefore, inhibiting the inflammatory response mediated by astrocytes can treat neuroinflammation after stroke. Moreover, recent studies have shown that inhibiting astrocyte-mediated inflammation can reduce cerebral I/R injury [48, 49]. Consistent with the results of these studies, the level of pro-inflammatory factors was up-regulated in our study when induced by OGD/R and MCAO/R, but their expressed were showed at lower levels after OXA treatment. These results indicate that OXA inhibits neuroinflammation by down-regulating the levels of pro-inflammatory factors, which is mediated by astrocytes.
Increasing evidences have reported that NF-κB signaling pathway is implicated in ischemic stroke [16, 50, 51]. As a pivot regulator of inflammation, NF-κB plays a key role in the inflammatory cascade . It regulates the transcription of downstream target genes, including TNF-α, IL-1β, IL-6 and iNOS, which promotes the expression and release of pro-inflammatory factors [18, 19], finally contributing to neuronal death, and exacerbating cerebral I/R injury [52, 53]. Therefore, the downregulation of NF-κB signaling pathway alleviates brain damage induced by I/R [16, 54]. The nuclear translocation of NF-κB subunit p65 is one of the indicators that can directly and accurately show that the NF-κB signaling pathway is activated . In this experiment, through the p65 and GFAP immunofluorescence images with U251 astroglioma cells subjected to OGD/R, we can clearly observe that the red fluorescence of p65 was significantly increased in the nucleus, while the fluorescence intensity of the cytoplasm was greatly reduced. The nucleoplasmic protein extraction and western blot analysis in U251 astroglioma cells after OGD/R and rat ischemic cortex were also consistent with it. These findings can fully explain that p65 was activated. However, treatment with OXA decreased nuclear translocation of p65 in U251 astrocytoma cells following OGD/R, indicating that OXA exerts anti-inflammatory effects by inhibiting NF-κB. In vivo experiments in MCAO/R rats also supported this, but it must be emphasized that the anti-inflammatory effect of OXA in vivo not only have affected astrocytes, but may have include microglia and neurons. Further work is necessary to illustrate this point. The anti-inflammatory effect of OXA can increase neuronal tolerance to ischemia and reduce neuronal damage in cerebral I/R injury.
MAPK signaling pathway is involved in inflammatory processes of cerebral I/R injury , and is the main signaling mechanism that regulates neuroinflammation . The activated MAPK causes the overproduction of pro-inflammatory factors . In contrast with ERK, which is responsible for cell survival and proliferation, the p38 is more closely related to the production of pro-inflammatory factors . In our study, the phosphorylation of ERK and p38 obviously increased at different reperfusion times in U251 astrocytoma cells after OGD/R. This indicates that the p-ERK and p38 signaling pathways are activated, and it can be inferred that the MAPK pathway in astrocytes is also involved in cerebral I/R injury. A similar phenomenon was observed in the MCAO/R rat. However, OXA inhibited the activation of p-ERK and p-p38. So, we have reason to infer that OXA reduces the production and release of pro-inflammatory factors by down-regulating the p-ERK and p-p38 signaling pathways.
To a certain extent, SB334867, an antagonist of OX1R, abolished the effect of OXA down-regulating the ERK and p38 pathways and inhibiting p65 nuclear translocation in OGD/R U251 astrocytoma cells. Our findings indicate that this anti-inflammatory effect of OXA is dependent on OX1R. However, it also needs to be verified in vivo. Therefore, there is an additional strong experimental evidence supporting OXA to function through OX1R .
Recent reports indicate that astrocyte apoptosis is involved in the pathological process of cerebral ischemia . In fact, astrocytes were the first to suffer an ischemic blow because their end feet directly surround the capillaries. Astrocytes play an important role in neuroprotection and survival of neurons, and its dysfunction seriously affects the survival ability of neurons. There is considerable evidence that neuron and astrocyte apoptosis coexist in cerebral ischemia and neurodegenerative diseases . Therefore, the study of regulating astrocyte apoptosis is particularly important. The excessive production of ROS in ischemic stroke causes the release of cytochrome C from the mitochondrial membrane into the cytoplasm , which activate caspase‑9 and consequently activate caspase‑3, DNA‑breaking enzymes and repair enzymes, leading to cell death [61–63]. The opening of mitochondrial permeability transition pores (PTP) is the key to the release of cytochrome C into the cytoplasm. PTP, which are channels across the inner and outer mitochondrial membranes, are also channels for the release of pro-apoptotic proteins including cytochrome C . Various factors, such as Ca2+, reactive oxygen species, adenine nucleotides, inorganic phosphate, etc., can induce the formation of PTP, which is enhanced by Bax and inhibited by Bcl-2 . Studies have reported that the ratio of Bcl‑2/Bax is reduced in the rat brain following cerebral I/R [66, 67]. In the cortical tissue of MCAO/R rats, the expression level of Bcl-2 was significantly decreased and the level of Bax was significantly increased, so that the ratio of Bcl-2/Bax decreased, which is consistent with the results of previous studies. Similarly, the ratio of Bcl-2/Bax was also reduced in OGD/R U251 glioma cells. However, after OXA treatment, the expression level of Bcl-2 increased while the expression level of Bax decreased, and the ratio of Bcl-2/Bax increased accordingly. Moreover, OXA inhibited the expression of cytochrome C, cleaved caspase-9 and cleaved caspase-3 induced by OGD/R in U251 glioma cells and MCAO/R in rat. In addition, Hoechst 33342 staining showed that OXA did reduced the rate of apoptosis caused by OGD/R. Therefore, from the above results, we infer that OXA alleviates astrocyte apoptosis by regulating the ratio of Bcl-2/Bax and inhibiting the expression of cytochrome C, cleaved caspase-9 and cleaved caspase-3. It still needs to be pointed out that the anti-apoptotic effects of OXA in vivo are multifaceted, which may include neurons, astrocytes, microglia and other nerve cells.