Liu et al. Journal of Neuroinflammation (2019) 16:8 https://doi.org/10.1186/s12974-019-1396-5 RESEARCH Open Access Mesenchymal stem cells alleviate the early brain injury of subarachnoid hemorrhage partly by suppression of Notch1-dependent neuroinflammation: involvement of Botch Wenchao Liu, Ran Li, Jian Yin, Shenquan Guo, Yunchang Chen, Haiyan Fan, Gancheng Li, Zhenjun Li, Xifeng Li, Xin Zhang, Xuying He and Chuanzhi Duan* Abstract Background: Activated microglia-mediated neuroinflammation has been regarded as an underlying key player in the pathogenesis of subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). The therapeutic potential of bone marrow mesenchymal stem cells (BMSCs) transplantation has been demonstrated in several brain injury models and is thought to involve modulation of the inflammatory response. The present study investigated the salutary effects of BMSCs on EBI after SAH and the potential mechanism mediated by Notch1 signaling pathway inhibition. Methods: The Sprague-Dawley rats SAH model was induced by endovascular perforation method. BMSCs (3× 6 10 cells) were transplanted intravenously into rats, and N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), a Notch1 activation inhibitor, and Notch1 small interfering RNA (siRNA) were injected intracerebroventricularly. The effects of BMSCs on EBI were assayed by neurological score, brain water content (BWC), blood-brain barrier (BBB) permeability, magnetic resonance imaging, hematoxylin and eosin staining, and Fluoro-Jade C staining. Immunofluorescence and immunohistochemistry staining, Western blotting, and quantitative real-time polymerase chain reaction were used to analyze various proteins and transcript levels. Pro-inflammatory cytokines were measured by enzyme-linked immunosorbent assay. Results: BMSCs treatment mitigated the neurobehavioral dysfunction, BWC and BBB disruption associated with EBI after SAH, reduced ionized calcium binding adapter molecule 1 and cluster of differentiation 68 staining and interleukin (IL)-1 beta, IL-6 and tumor necrosis factor alpha expression in the left hemisphere but concurrently increased IL-10 expression. DAPT or Notch1 siRNA administration reduced Notch1 signaling pathway activation following SAH, ameliorated neurobehavioral impairments, and BBB disruption; increased BWC and neuronal degeneration; and inhibited activation of microglia and production of pro-inflammatory factors. The augmentation of Notch1 signal pathway agents and phosphorylation of nuclear factor-κB after SAH were suppressed by BMSCs but the levels of Botch were upregulated in the ipsilateral hemisphere. Botch knockdown in BMSCs abrogated the protective effects of BMSCs treatment on EBI and the suppressive effects of BMSCs on Notch1 expression. (Continued on next page) * Correspondence: [email protected] Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, Guangzhou 510282, China © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Journal of Neuroinflammation (2019) 16:8 Page 2 of 20 (Continued from previous page) Conclusions: BMSCs treatment alleviated neurobehavioral impairments and the inflammatory response in EBI after SAH; these effects may be attributed to Botch upregulation in brain tissue, which subsequently inhibited the Notch1 signaling pathway. Keywords: Subarachnoid hemorrhage, Mesenchymal stem cells, Early brain injury, Neuroinflammation, Notch1, Botch Background [28–30]. Upon binding to its ligands Delta-1 or Jagged-1, Subarachnoid hemorrhage (SAH) represents a life- the Notch1 receptor is cleaved by disintegrin-metallopro- threatening cerebrovascular disease with high mortality tease tumor necrosis factor (TNF)-α-converting enzyme and morbidity rates [1, 2]. Although considerable efforts and γ-secretase complex, which releases the Notch1 intra- have shed light on the mechanisms involved in patho- cellular receptor domain (NICD) from the membrane logical events associated with SAH, the potential patho- [31]. The NICD then translocates to the nucleus, where it physiological mechanisms are still poorly understood. interacts with the transcription factor recombining bind- Angiographic vasospasm and delayed ischemic neuro- ing protein suppressor of hairless (RBP-Jκ), as wells as logical deficit were once regarded as the major cause of other regulators to modulate a variety of cellular metabo- the unfavorable prognosis after SAH [3]; however, clin- lisms, such as cell proliferation and differentiation [32, ical trials revealed that the poor prognosis associated 33]. In addition, increasing evidence indicates that Notch1 with SAH was not improved after use of anti-vasospastic signaling may also play an essential role in activation of drugs [4, 5]. Recent advances in SAH research demon- the inflammatory response in various diseases [34, 35]. Re- strated that early brain injury (EBI), which refers to cent studies demonstrated that Notch1 signaling not only whole brain injury within the first 72 h post-SAH, was contributed to activated microglia-mediated neuroinflam- deemed as one of the major causes of mortality and un- mation [36–38] but also aggravated neuronal degeneration favorable outcomes in SAH patients [6, 7]. As reported, [39], at least in part, via the nuclear factor-κB(NF-κB) sig- multiple physiological abnormalities during EBI trigger naling pathway during CNS diseases [40, 41]. MSCs trans- the activation of inflammatory cascades, which result in plantation has been demonstrated to suppress Notch1 blood-brain barrier (BBB) disruption and neuronal de- signaling in ischemic stroke, inflammatory bowel disease, generation [8, 9]. Therefore, it is critical to explore the and lupus [42–44]. However, whether MSCs administra- mechanisms of neuroinflammation and to discover ef- tion inhibits the Notch1 signaling pathway in EBI after fective therapies targeting the neuroinflammatory pro- SAH is unclear. cesses in SAH [10, 11]. Cation transport regulator homolog 1 (Chac1) was Mesenchymal stem cells (MSCs) have widely been used identified as neuroprotective gene 7 (NPG7), recently in experimental and clinical studies and have been dem- renamed Botch-using functional cloning strategy [45]. onstrated to have therapeutic potential in various system As an endogenous blocker of Notch1 receptor, Botch in- diseases such as ischemic stroke [12, 13], preterm brain hibits the S1 furin-like cleavage of Notch1, thus promot- injury [14], multiple sclerosis [15, 16], cardiac disease [17], ing maintenance of full-length Notch1 [46]. Recent and traumatic brain injury [18]. Exogenously transplanted studies reported that Botch promoted neurogenesis MSCs migrated to damaged tissue sites, where they differ- through antagonizing the maturation of Notch1 during entiated into several different lost or injured cell types and neocortical development and intracerebral hemorrhage modulate immune responses [19, 20]. However, increas- [47, 48]. However, whether Botch participates in the sup- ingly, recent evidence suggests that the protective effects pressive effects of BMSCs on Notch1 expression remains of MSCs are mediated in a paracrine manner [21, 22]. It unexplored. Thus, we hypothesized that BMSCs treat- has been reported that MSCs administration inhibited ment may alleviate the inflammatory response and neu- microglia activation in several central nervous system rodegeneration via inhibiting the Notch1 signaling (CNS) diseases [23, 24]. Although the beneficial effects of pathway in EBI after SAH. We also investigated whether bone marrow mesenchymal stem cells (BMSCs) trans- BMSCs treatment inhibited Notch1 activation in EBI plantation at 14 days [25, 26] and at 21 days [27] after SAH through a mechanism involving the upregula- post-SAH have previously been reported, to date and to tion of Botch in brain tissue. the best of our knowledge, no studies have investigated the effects of BMSCs treatment on EBI after SAH or the Methods possible underlying mechanisms. Animals The Notch1 signaling pathway plays an essential role in Adult male Sprague-Dawley rats (280–320 g) were pur- regulating cell fate decisions and in tissue homeostasis chased from the Animal Experiment Center of Southern Liu et al. Journal of Neuroinflammation (2019) 16:8 Page 3 of 20 Medical University (Guangzhou, China). The animals group (n = 24), SAH 24 h group (n = 31), SAH 24 h + were bred and housed in controlled temperature and hu- dimethylsulfoxide (DMSO) group (n = 31), and SAH midity conditions (22 ± 1 °C; 40–60%) with a 12:12 dark/ 24 h + DAPT group (n = 29). light cycle (lights on at 07:00 h; off at 21:00 h), and food and water were available ad libitum. All experimental Experiment 4 procedures were approved by the Southern Medical To assess the impact of knockdown of endogenous