The Experimental Research On The Potential Role Of Cerebral Cortical CD24 In Non-infectious Inflammatory Response After Traumatic Brain Injury

Backgroud: Increasing evidence indicates that non-infectious inflammatory response contributes to secondary brain injury following traumatic brain injury (TBI). However,the specific mechanisms of the signaling transduction and regulation remain largely unknown. We previously found that nuclear factor kappa B (NF-κB) dependent pathway could be activated to a peak in brain following TBI, and then it descended gradually. These findings suggested that some important factors or pathways could be activated to negatively regulate the non-infectious inflammatory response. Hence, it is becoming more and more important to find this kind of factor to inhibit the inflammatory response after TBI. Recently, it is reported that CD24 could down-regulate the activity of NF-κB, thus negatively regulating non-infectious inflammatory response. Moreover, the inflammatory response in brain after TBI is non-infectious. However, it remains obscure, as is whether CD24 plays a role in non-infectious inflammatory response after TBI. Hence, the aim of the current study is to explore the cerebral cortical expression pattern of CD24 and its potential role in non-infectious inflammatory response following TBI.Methods: Contused brain tissue biopsies were obtained from the pericontusinal cortex in TBI patients undergoing surgery, and samples of control group were from patients in the pathway during surgical removal of deep benign tumors. The expression and distribution of CD24 and myeloid differentiation primary response protein 88 (MyD88),a key regulator in inflammatory response,was assessed by quantitative real-time polymerase chain reaction (qPCR),Western blotting,immunohistochemistry and immunofluorescent double staining. The messenger RNA(mRNA) levels of tumor necrosis factor-a (TNF-a) and interleukin 1β (IL-1β),downstream pro-inflammatory cytokines of NF-κB dependent pathway, were measured by qPCR. Meanwhile, the outcomes of these patients were also investigated.Then we established an experimental TBI model in C57BL/6 mice using a modified Feeney’s model, and the expression and distribution of CD24 was detected by qPCR,Western blotting,and immunofluorescent double staining. According to the results in vivo, we treated mouse primary cortical neurons and astrocytes with recombinant high mobility group box 1 (rHMGB1) to simulate a neuronal inflammation model in vitro, and the expression and distribution of CD24 was measured by qPCR,Western blotting,and immunofluorescent staining. Based on the expression pattern of CD24, RNA interference was used to down-regulate the expression of CD24 both in vivo and in vitro. We detected the mRNA and protein levels of CD24 expression using qPCR and Western blotting; and NF-κB DNA-binding activity was assessed by electrophoretic mobility shift assay (EMSA);and mRNA levels of TNF-α and IL-1β were measured by qPCR. Thus, the specific effects of CD24 on inflammatory response were investigated after TBI.Results: The clinical study indicated that both the mRNA and protein levels of CD24 were markedly elevated after TBI, showing a time-dependent manner. The expression of CD24 was gradually enhanced with the extension of time after TBI, and it could be detected in neurons,astrocytes,and microglia. In animal study,we also found a higher level of CD24 mRNA and protein expression in TBI groups as compared to that in sham group, with the peak on day 7, and the elevation of CD24 could be detected in neurons, astrocytes, and microglia. Meanwhile, down-regulation of CD24 with RNA interference could induce a significant increase of NF-κB DNA-binding activity and mRNA levels of TNF-a and IL-1β. In vitro experiments, application of rHMGB1 could induce the activation of NF-κB dependent inflammatory pathway both in mouse primary cortical neurons and in astrocytes. Meanwhile, CD24 mRNA and protein expressions climbed to a peak level at 36 h after rHMGB1 addition. Similar to the animal study, NF-κB dependent inflammatory pathway could be significantly up-regulated with the RNA interference to CD24 both in neurons and in astrocytes.Conclusion: We first reported that CD24 could be markedly elevated in the brain following traumatic brain injury in patients, which was demonstrated by experiments both in vivo and in vitro. Meanwhile, down-regulation of CD24 with RNA interference could significantly up-regulate the activity of NF-κB dependent inflammatory pathway. Our findings suggested that the elevation of cortical CD24 might negatively regulate the non-infectious inflammatory response during secondary brain injury after TBI, thus providing a novel target for therapeutic intervention of TBI.