Potential Mechanism Of Curcumin In Inhibiting Glial Scar Formation After Spinal Cord Injury And Its Effect On Nerve Regeneration

BackgroudSpinal cord injury(SCI) can result in irreversible neurological damage, for which effective treatments are lacking. This type of injury represents a great challenge in human health. The abortive attempts of axon regeneration after spinal cord injury are influenced by glial scar formation, inflammatory cell activation and the production of inhibitory extracellular matrix. Activated astrocytes by inflammatory cytokines to form reactive gliosis and lead to the glial scar formation. Glial scar also can secrete pro-inflammatory cytokines and extracellular matrix(ECM) components, aggravate the secondary injury, lead to a vicious cycle. Hence, suppressed glial scar formation is an important strategy for improved the neural functional recovery of SCI.Curcumin(cur), a prominent yellow pigment and major active component of turmeric, is extracted from powdered dry curcuma Longa rhizome and has been used for centuries as a spice and food coloring. Extensive studies over recent decades have demonstrated that cur has a variety of therapeutic activities due to its potent antioxidant, anti-inflammatory, anti-fibrotic and anti-tumor properties, which are mediated by numerous molecular targets, including transcription factors, growth factors, receptors, cytokines, chemokines, and enzymes. There have been reports that cur can inhibit glial scar formation, but its mechanism of action and targets still need further research.Inflammation plays a key role in secondary spinal cord injury. As resident immune cells in central nervous system, microglia are activated after SCI and can release large quantities of inflammatory cytokines, which are important element of the mediated inflammatory response after SCI. Inhibited the activity of microglia can significantly promote motor functional recovery after spinal cord injury. Cur can suppress the microglia activity after traumatic brain injury, but its role in the microglia activation after SCI lack of systematic research. Inflammatory reaction is an important factor affecting neural stem/progenitor cells(NSPCs) proliferation and differentiation. Inhibited Inflammation can promote neurogenesis and oligodendrogenesis from NSPC differentiation.Based on the above theory, we make the following assumptions: Cur could inhibit astrocyte and microglia induced inflammation after SCI. It could improve the motor function recovery through potent anti-inflammatory property. Control of the inflammation post SCI could improve the internal environment of NSPCs and it could play a positive role in NSPCs differentiation. This research focus on the several aspects.Part 1: Curcumin inhibits glial scar formation after SCIBackground:SCI can activate glial cells and lead to reactive astrogliosis, which can severely reduce the space for nerve growth. Glial cells can secrete a large amount of extracellular inhibitory components, thus altering the microenvironment of axon growth. Both of these factors seriously impede nerve regeneration. In the current study, we established a rat model of SCI and treated the animals with different concentrations of cur to investigate the role of cur in glial scar formation after SCI.Materials and methods:1. The SD rats were used to establish SCI model and treated the animals with different concentrations of cur(300mg/kg, 100 mg/kg, 30 mg/kg) for 7 days. Methyl prednisolone treatment was used as positive control group.2. At 8 weeks post SCI, the BBB scoring, inclined plane test and BDA tracing of nerve fiber bundles were used to assess the motor function recovery.3. At 8 weeks post SCI, the HE staining and MRI were used to detect the cystic cavities volume.4. At 2 weeks post SCI, we used the immunohistochemical analysis to observe the expression of both intracellular and extracellular components GFAP and CSPG.5. At 2 weeks post SCI, we used the ELISA and PCR to detect the expression of inflammatory cytokines in glial scar.Results:1. The BBB scoring, inclined plane test and BDA staining in cur treatment group was significantly higher than those of in the MP treatment group and the sham group. And the effect was positively correlated with the cur concentration.2. Cur could inhibit glial scar formation 8 weeks post SCI.3. Cur could reduce the expression of intracellular components(GFAP) and extracellular components(CSPG) in glial scar.4. Cur could suppress expression of inflammatory cytokines in glial scar.Conclusion:Cur is beneficial for morphology and motor function recovery in rats after SCI. Cur suppresses the expression of intracellular intermediate filament protein GFAP of glial scar through anti-inflammation. Cur reduces the deposition of ECM components CSPG by inhibiting transcription factors. The joint inhibition of the intra- and extra-cellular components is the mechanism by which cur inhibits glial scar formation.Part 2: Effect of Curcumin on astrocyte activated and potential mechanismBackground:Using both in vivo and in vitro experiments, the current study investigated the phenotypic transformation of astrocytes following cur intervention during the processes of inflammation and fibrosis and determined details of the relationship between cur treatment and the glial scar components GFAP and CSPG.Materials and methods:1. The SD rats were used to establish a model of SCI and treated with 300mg/kg of cur for 7 days. DMSO treatment was used as control group.2. We used cultured astrocytes to establish a reactive astrogliosis model in vitro, and then interfered the astrocytes with NF-κb subunit p65 si RNA and SOX9 siRNA. EMSA、WB and PCR were used to detect the transfection efficiency.3. We detected the relationship between glial scar intracellular components GFAP and NF-κb signaling pathways using immunofluorescence, WB and PCR. We also observed the chemokine released by astrocytes with ELISA and detected the number of immune cells in glial scar with Flow Cytometric Analysis.4. We detected the relationship between glial scar extracellular components CSPG and SOX9 signaling pathways and the expression of fibrosis related molecular α-SMA with immunofluorescence, WB and PCR.Results:1. Cur treatment significantly decreased expression levels of NF-κb signaling pathway p-IKK-α, p-IKK-β, p-IκB-α and p65 and increased the expression level of IκB-α, thus down-regulating the expression of the astrocyte intermediate filament protein GFAP.2. Cur inhibited activated astrocytes expressing chemokine MCP-1, RANTES and CXCL10 via NF-κb signaling pathway.3. Cur inhibited macrophage and T-cell infiltration in glial scar via NF-κb signaling pathway.4. Cur inhibited SOX9 mediated ECM deposition。5. Cur inhibited SOX9 mediated α-SMA expressionConclusion:This study demonstrates that cur could inhibit intra- and extra-cellular components of glial scars via its dual effects. Cur reduces infiltrations of chemokine-induced macrophage and T-cells via the NF-κb pathway. Meanwhile, Cur suppresses astrocyte α-SMA expression via the SOX9 pathway. Cur possesses simultaneous anti-inflammatory and anti-fibrotic functions. These advantages give cur enormous potential as a drug for SCI treatment, providing important information for studies on the dual-target regulation of intracellular and extracellular glial scar components.Part 3: Effect of Curcumin on microglia activated and potential mechanismBackground:In the current study, we established a mice SCI model in vivo and induced primary microglia activated with LPS in vitro by using Nrf2(+/+) and Nrf2(-/-) mice and treated with different concentrations of cur. Using both in vivo and in vitro experiments, we investigated the role of cur in microglia activated and potential mechanism.Materials and methods:1. The Nrf2(+/+) and Nrf2(-/-) mice were used to establish the SCI model and treated the animals with different concentrations of cur(100mg/kg, 50mg/kg, 25mg/kg).2. We detected the relationship between the microglia activity and the expression of Nrf2 by using WB analysis 28 days post SCI.3. With the Nrf2(+/+) and Nrf2(-/-) microglia in vitro, we used cur or ERK inhibitor U0126 to intervene the LPS-induced primary microglia.4. We detected the relationship between microglia activity and the expression of Nrf2 at 7 days post SCI using immunofluorescence, WB, PCR and Flow Cytometric Analysis.5. WB analysis was used to detect the expression of ERK, stat3 and p65.Results:1. The expression of Nrf2 and iNOS were rapidly up-regulation and peaked at 7 days post SCI.2. Cur translocated Nrf2 from the cytoplasm into the nucleus and activated it. Cur inhibited microglia activity and reduced iNOS expression. And the activated effect was positively correlated with the cur concentration. In addition, Nrf2 gene knockout could abolish the inhibition of microglia by cur.3. Compares with LPS Nrf2(+/+) group, the expression of p-ERK and iNOS in LPS+cur Nrf2(+/+) microglia were decreased, whereas there was no statistically significant difference between the LPS Nrf2(-/-) group and LPS+cur Nrf2(-/-) group.4. ERK inhibitor U0126 suppressed ERK phosphorylation and reduced p-stat3 and p-p65 expression in Nrf2(-/-) microglia.5. Cur regulated the Nrf2/ERK/p65 signaling pathway in microglia.Conclusion:Cur could inhibit microglia activated and improve the motor function recovery through suppressing microglia induced inflammation. Cur inhibited microglia activated via Nrf2/ERK/p65 signaling pathway.Part 4: Effect of suppressing inflammation on neural stem/progenitor cells differentiation and potential mechanismBackground:Neural stem/progenitor cells(NSPCs) are an important source of cells for cell replacement therapy after SCI. How to induce oriented differentiation of NSPCs towards neurons and oligodendrocytes is a challenging point in neuroscience research. In the present study, we cultured microglia in vitro and polarized them into M1 and M2 phenotypes, used different microglia supernatants to induce NSPCs differentiation, and elaborated the effects of different microglia phenotypes on NSPCs differentiation and their mechanisms.Materials and methods:1. After interfered primary microglia to different polarization statuses, we researched the expression levels of M1 subtype markers i NOS and CD86 and M2 subtype markers CD206 and Arg1 with immunofluorescence assay, WB and PCR.2. We collect the supernatants from different polarization of microglia and use as the culture media for induced NSPCs differentiation. Immunofluorescence assay and WB were used to detect the ratios of neurons, oligodendrocytes and astrocytes generated by NSPC differentiation.3. We detected the expression of PPARγ and 15d-PGJ2 in M2 microglia with LC—MS analysis and ELISA.4. We used the immunofluorescence assay and WB to observed differentiation of NSPCs with PPARγ inhibitor GW9662 intervention.5. We detected the expression of PPARγ and β-catenin in NSPCs differentiation with WB analysis.Results:1. LPS+IFN-γ polarized microglia phenotype to M1, IL-4 polarized microglia phenotype to M2.2. M2 microglia supernatant increased the numbers of neurons and oligodendrocytes generated by NSPCs differentiation, while reducing the differentiation ratio of astrocytes.3. As compared with M1 microglia, M2 microglia had a higher expression level of PPARγ and 15d-PGJ2, and the difference was statistically significant.4. Using GW9662 to intervene PPARγ, which is the receptor for 15d-PGJ2, could promote NSPCs differentiation towards astrocytes, and inhibit the differentiation ratios of neurons and oligodendrocytes.5. M2 microglia supernatant could increase the expression of PPARγ and β-catenin in NSPCs.Conclusion:The present study found that M2 microglia can increase the ratios of neurons and oligodendrocytes generated by NSPC differentiation, and decrease the number of differentiated astrocytes. M2 microglia promote NSPC differentiation by activating the PPARγ/β-catenin signaling pathway. Our experiment provides further experimental support and alternative options for promoting NSPCs oriented differentiation.In summary, curcumin inhibits astrocytes and microglia mediated inflammatory response, at the same time also can inhibit astrocytes fibrosis. Cur reduces the expression of intracellular and extracellular glial scar components through dual-target regulating both inflammation and fibrosis after SCI in the rat. Curcumin can favorable neural stem cells to differentiate into neurons and oligodendrocytes via suppressing inflammation and promote the neural functional recovery after spinal cord injury.