MP Resulting In Autophagic Cell Death Of Microglia Through Zinc Changes Against Spinal Cord Injury

ObjectiveThis study sought to investigate the exact effect of MP on SCI, and its mechanism.MethodsWe examined the effect of MP in a model of SCI both in vivo and in vitro. 110 SD femal adult rats were randomly divided into 2 groups, and in each groups, subgroups were set up, 5 rats in each subgroups. The model of SCI was established by using the modified Allen’s method on T10 of spinal cord. BBB scores were analyzed at different time points(normal, 3D, 7D, 21 D, 60D). The effect of MP on the numbers of motor neurons in spinal cord was investigated using Nissl staining at 12 h after SCI. Protein Iba-1 of microglia was assessed by Immunohistochemistry at 12 h and 18 h after SCI. In SCI rat, atomic absorption spectrometry was used to quantify the content of zinc elements of spinal cord tissue and serum. In vitro, cell viability were tested under different stress by MTT. Then we visualized morphology of microglia in each groups by immunofluorescence staining of Iba-1. And we assessed TNF α, IL 6 and IL 10 cytokine secretion levels in microglia culture medium by ELISA. Then we detected i NOS, LC 3B, ZIP8, NF-κβ, Beclin-1 protein by western blot. Finally, we examined cellular labile zinc changes through cells stained with Fluo Zin?-3 AM.ResultsWe found that administration of MP produced an increase in the Basso, Beattie and Bresnahan scores and motor neurons counts of injured rats. Besides the number of activated microglia was apparently reduced by MP in vivo, and Beclin-1 dependent autophagic cell death of microglia was induced by MP in LPS induced model. Furthermore, relevant markers associated with activation of microglia, which could produce a neuroinflammatory environment, were decreased by MP administration. At the same time, MP increases cellular zinc concentration and level of ZIP8, and TPEN could revert effect of MP on autophagic cell death of microglia. Finally, we have found that MP could inhibit NF-κβ in LPS induced model.ConclusionsThese results shows that the MP could result in autophagic cell death of microglia, which mainly depends on increasing cellular labile zinc, and may associated with inhibition of NF-κβ, and that MP can produce neuroprotective effect in SCI.