Roles Of 5-lipoxygenase And Cysteinyl Leukotriene Receptors In Oxygen-glucose Deprivation-induced Injury In Rat Astrocytes

5-Lipoxygenase (5-LOX) is a key enzyme metabolizing arachidonic acid (AA) to form inflammatory mediators, leukotriene B₄ (LTB₄) and cysteinyl leukotrienes (CysLTs, including LTC₄, LTD₄ and LTE₄). CysLTs are potent inflammatory mediators and involved in various diseases. The actions of CysLTs are mediated via activating their receptors (CysLT₁ and CysLT₂ receptors). In recent studies, 5-LOX, CysLT₁ and CysLT₂ receptors are reported to express in the brain, and they may be involved in the pathogenesis of various CNS diseases, such as cerebral ischemia, trauma, tumor, epilepsy and aging. After cerebral ischemia, both the expression and activity of 5-LOX in the brain are increased, and the contents of CysLTs are elevated in the brain tissue. Furthermore, the expression of CysLT₁ and CysLT₂ receptors are induced after cerebral ischemia. Inhibitors of 5-LOX, such as AA861, MK-886 and nordihydroguaiaretic acid, exert protective effects on cerebral ischemia. Our previous studies have also shown the protective effect of CysLT₁ receptor antagonists, including pranlukast (ONO-1078) and montelukast, on neuronal injuries in the acute phase and astrogliosis/glial scar formation in the late phases after focal cerebral ischemia.However, lots of questions should be investigated to clarify the involvement of 5-LOX, CysLT₁ and CysLT₂ receptors in brain ischemic injury. For example, are 5-LOX, CysLT₁ and CysLT₂ receptors expressed in astrocytes? Are they involved in oxygen-glucose deprivation (OGD)-induced ischemic injury in astrocytes? If they are involved, how do they play their roles in the ischemic injury mediated via different CysLT receptor subtypes?Therefore, in the present study, we want to answer the above questions using the OGD-induced in vitro ischemic injury model in the cultured rat primary astrocytes.1. 5-LOX pathway is involved in oxygen-glucose deprivation-induced injury in rat astrocytesMembrane phospholipids are catalyzed to form arachidonic acid (AA) by activated phospholipase A₂ (PLA₂). Free AA is metabolized by two major enzyme systems, cyclooxygenase (COX) and 5-LOX. To determine whether the COX pathway or 5-LOX pathway is involved in OGD-induced injury in rat astrocytes, the effects of following agents on OGD-induced injury were observed in rat astrocytes, i.e., PLA₂ inhibitor (quinacrine), COX inhibitor (indomethacin), 5-LOX inhibitor (caffeic acid and zileuton), 5-LOX activating protein (FLAP) inhibitor (MK-886). The results showed that the proliferation of astrocytes was significantly increased after 1-h OGD and 48-h recovery; astrocyte viability was significantly decreased by about 50% indicating cell injury) after 4-h OGD and 24-h to 72-h recovery as detected by MTT reduction assay. A PLA₂ inhibitor quinacrine (1 and 5 μM), an FLAP inhibitor MK-886 (2 and 10 μM) and 5-LOX inhibitors caffeic acid (5 and 25 μM) and zileuton (1 and 5 μM) concentration-dependently ameliorated OGD-reduced astrocyte proliferation or cell injury, while a non-specific COX inhibitor indomethacin (1-25 μM) did not show this effect. After OGD for 1 or 4 h, the release of CysLTs from astrocytes was increased; the release was positively related to the treatment time of OGD. The OGD-increased release of CysLTs was inhibited by caffeic acid (5μM),zileuton (5 μM) and MK-886 (1 μM). These results indicate that 5-LOX pathway is involved in OGD-induced proliferation and injury in astrocytes; the products of 5-LOX (CysLTs) may mediate OGD-induced proliferation and injury via CysLT receptors in astrocytes.2. CysLT₁ and CysLT₁ receptors mediate OGD-induced injury in rat astrocytesIn the above experiments, it has been clarified that 5-LOX is activated to produce more CysLTs after OGD in rat astrocytes. Furthermore, in previous studies of our laboratory, we have demonstrated that CysLT₁ receptor antagonists (pranlukast and montelukast) exert the neuroprotective effect on cerebral ischemia, and inhibit astrogliosis and glial scar formation in the boundary zone around the ischemic core after focal cerebral ischemia. These findings strongly suggest that CysLT₁ and CysLT₂ receptors may modulate OGD-induced changes in astrocytes. Therefore, in the present experiments, we determined whether CysLT₁ and CysLT₂ receptors are expressed in astrocytes, and what changes occur in the receptor expressions and responses after OGD treatment. The results of RT-PCR indicated that CysLT₁ receptor mRNA was moderately expressed, but CysLT₂ receptor was much weakly expressed in rat astrocytes. The expression of CysLT₁ receptor mRNA was increased after 1-h OGD; while the expression of CysLT₂ receptor mRNA was increased after 4-h OGD. The pharmacological experiments showed that 1-h OGD-induced astrocyte proliferation was inhibited by 1～5μM montelukast (the selective antagonist of CysLT₁ receptor) and 1-5μM BAY u9773 (a non-selective antagonist of CysLT₁/CysLT₂ receptor), but not by 0.2-5 μM AP-100984 (the selective antagonist of CysLT₂ receptor). Conversely, 4-h OGD-induced astrocyte injury was inhibited by AP-100984 (1-5 μM) and BAY u9773 (1-5 μM), but not by montelukast (0.2-5 μM). On the other hand, LTD₄, the exogenous agonist of both CysLT₁ and CysLT₂ receptors, induced astrocyte proliferation at a lower concentration (1 nM), butdamaged astrocytes at a higher concentration (100 nM). Montelukast (1-5 μM) and BAY u9773 (1-5 μM) inhibited the low-concentration LTD₄-induced astrocyte proliferation, while AP-100984 (1-5 μM) and BAY u9773 (1-5 μM) inhibited the high-concentration LTD₄-induced astrocyte injury. From these results, we conclude that both low-intensity OGD and low-concentration LTD4 induce CysLT₁ receptor-mediated astrocyte proliferation, while both high-intensity OGD and high-concentration LTD₄ induce CysLT₂ receptor-mediated astrocyte injury.Conclusions1. 5-LOX pathway is involved in OGD-induced injury in rat astrocytes; inhibition of key enzyme activities in this pathway can produce protection against OGD-induced astrocyte injury. OGD can intensity-dependently activate 5-LOX in astrocytes and lead to release of CysLTs.2. CysLTi receptor is moderately expressed, but CysLT₂ receptor was weakly expressed in rat astrocytes. Low-intensity OGD increases the expression of CysLT₁ receptor, while high-intensity OGD and high-concentration LTD₄ induce the expression of CysLT₂ receptor in astrocytes.3. Low-intensity OGD or low-concentration LTD₄ induces the CysLT₁ receptor-mediated astrocyte proliferation; while high-intensity OGD or high-concentration LTD₄ induces the CysLT₂ receptor-mediated astrocyte injury.4. In summary, the in vitro ischemic injury (OGD) can activate 5-LOX, and increases synthesis and release of CysLTs; the released CysLTs induce CysLT₁ receptor-mediated astrocyte proliferation and CysLT₂ receptor-mediated astrocyte injury.