| ObjectivesWith the development of economic and an aging risk factors of the population, centralnervous system disorders such as hypertension, neurodegenerative disorders, trauma andother factors have increased year by year, the incidence of CNS injury showing anincreasing trend, which has a very high rate of death and disability, give each family atremendous economic pressure and psychological burden. Although the neurosciencecommunity around the world invest a lot of energy to carry out related research for suchCNS diseases, but how to activate endogenous neural stem cells and promote thedifferentiation of neural stem cells into neurons after the nervous system injury, yet noexact effective method that truly become treatment through clinical trials in patients whowith damage of the central nervous system. Therefore, we explore the use of curcumionintervention to activate neural stem cells after CNS injury.CNS (central nervous system, CNS) injury can cause severe neurological dysfunction,bring a heavy financial burden to the family and the society. Currently, surgery, gene therapyand rehabilitation methods have been used for the treatment of central nervous system injury,although these means have some advantages, most patients still can not obtain a substantialrecovery of function. Thus, effective treatment of CNS injury are urgent. After CNS injury,neural stem cells in the damaged region (neural stem cells, NSCs) can be proliferate anddifferentiated into three kind of neuron cells, the differentiation ratio of neurons, to a largeextent determines the prognosis of patients with CNS injury.Traditional Chinese medicine (TCM) turmeric are widely used in India and Chinesetraditional medicine. Curcumin is multiple phenolic substances which extract from turmeric,with a variety of biological activity and almost no side effects, the key of clinical applicationof curcumin is to elucidate the mechanism of action. The large amount of research before hasproved the curcumin has the nerve protective effect. Kim, found that curcumin has the effectof neuroprotective and its optimal concentration is500nmol/L. Wnt protein is a kind of secretion widespread from polyps to human, the activation ofWnt/β-catenin signaling pathway can significantly promote neural stem cells differentiationproportion to neurons in the hippocampus of adult animals, the regulation of Wnt/β-cateninsignaling pathway play a critical role in nerve regeneration in the brain, it has been clear thatwnt3a, β-catenin belong to classic Wnt/β-catenin signaling pathways, and play animportant role in neural development.At present, there is no literature reported to clarify the relationship between curcuminand the classic Wnt/β-catenin signaling pathway in neural stem cells. This experiment wasdesigned to observe the change of wnt3a and β-catenin in each intervention groups ofneural stem cells. Western blot and RT-PCR methods were performed to detect the change ofwnt3a、β-catenin protein and wnt3a、β-catenin mRNA expression level in Wnt signalingpathways, in order to clarify the relationship of curcumin and classic Wnt/β-cateninsignaling pathway. This study tries to reveal the possible mechanism of neuroprotectioneffect of curcumin, and provide experimental basis for neural protection treatment.Materials and methodsUsing Sprague-Dawley (SD) rats at a gestational age of14.5days, we isolated neuralstem cells from the two-thirds of the fetal rat anterior brain. The neural stem cells werepassaged three times using the half media replacement method and identified using cellularimmunofluorescence. After continuous passaging for three generations, we cultured cells inmedia without bFGF and EGF. Then, we treated cells in five different ways, including ablank control group, a group treated with IWR1(10μ mol/L), a group treated withcurcumin(500nmol/L), a group treated with IWR1+curcumin and a group treated withDMSO(10μmol/L). We then measured the protein and RNA expression levels for wnt3aand β-catenin using western blotting and RT-PCR.Results1. The extraction of neural stem cells, subculture and identification(1) Using Sprague-Dawley (SD) rats at a gestational age of14.5days as the researchobject, extract the embryonic neural stem cells and puts it into DMEM/F12which adding2%B27and20ng/ml of bFGF、20ng/ml EGF for culturing, we found that the ball diameterincreased obviously when the second generation of neural stem cells was trained.(2) The subculture of neural stem cells: we used pancreatic enzyme at the concentration of1.25%digestion to extend the neural stem cells, the effect is better thatcombination of machinery and percussion batches.(3) The identification of neural stem cells: Immunochemical fluorescent dye confirmedthat nestin positive was NSCs cells, after Induced by the serum broth some cells presentMAP2positive, few part of cells present GFAP positive cells.2. After continuous cultivation for three generations of neural stem cells, five groupswere designed, including: blank control group, the IWR-1(Wnt signaling pathwaysspecific blocker10mu mol/L) treatment group, curcumin500nmol/L, IWR-1+curcuminIWR-500nmol/L group (add IWR–1first, add the curcumin20min after), DMSO group.After culturing for3days, immunofluorescence cell technology is carried out, we found thatIWR-1group reduce the proportion of NSCs differentiation into neurons, however thecurcumin group increased the differentiation of neural stem cells into neurons, the ratio thatIWR-1combined curcumin group was decreased, compared to the blank control group andDMSO group.3. Western-blotting：After the third generation of cells had been treated for72h,weobserved that wnt3a and β-catenin expression was significantly increased in the groupreceiving500nmol/L curcumin but not in the other groups. Furthermore, cells in theIWR1-treated group showed decreased wnt3a and β-catenin expression, and wnt3a andβ-catenin was also decreased in the IWR1+500nmol/L curcumin group. No obviouschange was observed in the DMSO group. RT-PCR: RT-PCR showed similar changes tothose observed with the western blotting experiments(P＜0.05).Conclusions1. After treated with curcumin, we found that the proportion of neural stem cells intoneurons obvious increased in the curcumin group,while the ratio was reduced in the IWR-1group and blank group.2. After treatment of IWR-1, the expression of wnt3a and β-catenin protein andmRNA which belongs to the Wnt/β-catenin signaling pathway were significantly reduced,and the expression of wnt3a and β-catenin in the curcumin group was increasedsignificantly, shows that curcumin can activate the Wnt/β-catenin signaling pathway. Theresults suggested that curcumin exhibits a neuroprotective effect through the classicalWNT/β-catenin signaling pathway 3. This study was tend to provide more theoretical basis of neuroprotective effect ofcurcumin in the central nervous system after injury, which provides the theoretical basis ofcurcumin in clinical research. |
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