Metallothionein-Ⅰ/Ⅱ Protect Astrocytes Against Oxygen Glucose Deprivation And Reperfusion Injury

Metallothioneins are ubiquitous low molecular weight, cysteine-rich and metalbinding proteins，and have multiple functions, such as detoxification of heavy metals,essential metal homeostasis regulating and protect against oxidative stress. There arefour types of MTs in mammals, including MT-I、MT-II、MT-III and MT-IV. The majorisoforms in CNS are MT-I、MT-II (MT-I/II) and MT-III, MT-I/II mainly locate inastrocytes and MT-III in neuron. In recent years, more and more studies show thatMT-I/II have important neuroprotective function in cerebral ischemia, but its specifictargets and mechanisms are unclear.Objective: The subject use MT-I/II knockout (MT-/-) mice and wild-type (MT+/+)mice to establish neonatal mice cortical primary cultured astrocytes, through culturedcells deprived of oxygen and glucose (Oxygen glucose deprivation, OGD), and thenre-oxygen and glucose--reperfusion (Reperfusion, Rep) to establishischemia-reperfusion model in vitro, to clarify the role of MT-I/II in astrocytes againstOGD/Rep induced injury and to explore the mechanism, and the role of MT-I/II inexogenous compound (resveratrol) mediated protection of astrocytes.Methods and results: We obtained primary cultured cortical astrocytes fromC57BL/OLA129MT+/+and-/-neonatal mice, and apply the astrocyte-specific protein(GFAP) to identify the cells’purity by immunocytochemistry method. The resultsshowed that cultured cells’ purity can reach more than90%, cell morphology and cellcounts showed that MT+/+and-/-astrocytes are basically similar in growth rate,morphology and other features, indicating that MT-I/II’s knockout did not significantly affect the structure and function of astrocytes in physiological conditions.The primary cultured MT+/+and-/-astrocytes were exposed to6h oxygen glucosedeprivation (OGD) and24h reperfusion(Rep) to evaluate the cell injury as indicatedby the morphology changes, cell viability,apoptosis and LDH leakage. we found thatOGD6h/Rep24h significantly induced cell injury as evidenced by nuclear shrinkage,vacuolization, apoptosis, cell death and higher LDH leakage in astrocytes;comparatively, the MT-/-astrocytes are more sensitive to OGD/Rep induced injury,indicating that MT-I/II protect astocytes against OGD/Rep injury.Resveratrol is a polyphenolic compound, recent studies have shown thatresveratrol has a significant protective effect in cerebral ischemic injury and itsmechanism may related to MT-I/II, but the specific mechanism is still unclear. Toconfirm the role of MT in resveratrol-mediated anti-ischemic brain injury, we addresveratrol to MT+/+and-/-primary cultured astrocytes, to clarify whether resveratrolexerts its neuroprotection by regulating MT. We firstly gave MT+/+astrocytesdifferent concentrations of resveratrol treatment (10nM,100nM,1μM,10μM) andOGD/Rep, select the appropriate concentration (10μM) by its morphological changesfor the following study. MT+/+and-/-astrocytes are treated with10μM resveratrol andOGD/Rep, vacuolization, apoptosis, cell death in MT+/+cells occur less thannon-resveratrol group in morphology. And LDH leakage decreased more thannon-resveratrol group, espacially in MT+/+astrocytes and Rep process(P<0.05), showprotective effect of resveratrol.We use fluorescense probe labeling cells to detect oxdative damages bymeasuring cells mitochondrial membrane potential and cells’ROS level afterOGD/Rep and Resveratrol treatment, the results show lower mitochondrial membranepotential and higher ROS level after OGD/Rep, means oxdative damages occurred,and MT-/-cells got more serious damages. After adding Resveratrol, cells had lessinjury espacially in MT+/+astrocytes.We set multiple time points (2h,6h,12h,24h) in reperfusion process, theimportant antioxidants Mn-SOD’s and MT’s mRNA expression levels were measured by RT-PCR. The results showed that the expression of MT-I and MT-II of MT+/+cellsboth significantly increased in Rep24h significantly, that means MT-I/II mRNAexpress and conduct protective role after Rep12h to24h. Comparing the SODexpression of MT+/+and-/-astrocytes, we see significant difference between them.MT+/+had a sudden increase at Rep24h,which same with MT-I/II. MT-/-’s SODincreased highly at Rep6h, and then decreased. MT-/-cells may combat oxidativestress by regulating the expression of SOD and other antioxidants to achieveself-protection due to the lack of this important antioxidant protein-MT.After addingprotective drugs resveratrol, MT expression increased at Rep6h time point,indicating that expression of MT was18h advanced compared to non-resveratrolgroup (at Rep24h increased significantly). MT+/+astrocytes’ SOD levels dropsignificantly after OGD treated with10μM resveratrol, suddenly rise at Rep6h, thendecrease, compared to non-resveratrol groups (increase at Rep24h), which isadvanced18h; MT-/-cells’ SOD immediately dropped to very low level after OGD,and return to normal levels at Rep2h, keeping to6h.Compared with non-resveratrolgroup, the time is advanced4h. From these results that resveratrol can exert aprotective effect by regulating the expression levels of SOD and MT-I/II, and moresignificantly in MT+/+astrocytes, also suggests the mechanism of anti-oxidative stressof resveratrol may be the regulation of MT-I/II.Conclusion: In summary, we establish oxygen-glucose deprivation reperfusion modelwith primary cultured astrocytes to investigate the role of MT-I/II in cerebral ischemiareperfusion injury. These models confirm MT-I/II have significant protective effectagainst OGD/Rep induced astrocytes’ injury, suggesting that its mechanism is closelyrelated to MnSOD; also confirmed that resveratrol may exert its anti-ischemic injuryfunction by regulating the expression MT-I/II.