| BackgroundSpinal cord injury(SCI)is a severe condition involving a variety of pathogenic factors that lead to structural and functional injury of the affected nerves,resulting in the loss of voluntary movements and sensation below the damaged plane.The SCI patients are often associated with poor quality of life,lower expectancy of life,and generating severe economic and social burdens on the their families and society.Due to the limited regenerative capability of neuronal elements and its negative prognosis,the treatment of SCI continues to represent a difficult medical challenge.Conventional treatment of SCI focuses on stabilizing the injured area via surgery,preventing secondary injury through pharmacological intervention,and rehabilitation to prevent loss of function and to help regain the loss functions.However,these treatments have limited success as they cannot stimulate spinal cord regeneration,developing an effective therapy for SCI remains an urgent clinical need.For the sake of preserving damaged tissue integrity and providing physical support and trophic supply for axon regeneration,cell-based therapy has come to the front stage in therapy for SCI with the constant progress of stem cell engineering.At present mesenchymal stem cells(MSCs)are considered a promising material to stimulate neuroregeneration,related to their high biosafety and immunomodulatory properties,their ability to synthesize neurotrophic factors.Adipose tissue-derived stromal cells(ADSCs)are a superior alternative to MSCs from other tissues because of their abundant availability and excellent expansion and proliferative capacities,ease of harvest.Safety and efficacy of ADSCs therapy in ameliorating SCI have been evaluated in in vitro and SCI in vivo.Compared to bone marrow mesenchymal stromal cells(BMSCs),ADSCs demonstrated a higher survival rate when transplanted into spinal cord injury sites in experimental SCI models in vivo.Studies also reported that ADSCs secreted cytokines and growth factors which improved axonal regeneration and reduced cavity formation.However,despite the evidence supporting the use of ADSCs for SCI treatment,the specific mechanisms by which ADSCs promote spinal cord injury repair are not fully clear.Torres-Espin et al.reported that MSCs transplantation after SCI enhanced the expression of procollagen-lysine,2-oxoglutarate 5-dioxygenase 2(PLOD2),an enzyme that contributes to extracellular matrix(ECM)organization by catalyzing the formation of collagen crosslinks.PLOD2 is regulated by HIF-1?,TGF-?1,and microRNA-26a/b through the PI3K-AKT or the TGF-?/Smad signaling pathways in tumor cells.However,the regulation and role of PLOD2 on SCI remain uncertain.Recent studies suggested that the therapeutic effect is mostly exerted by their paracrine activity as ADSCs have been found to secrete a broad range of bioactive molecules.As an important factor secreted by ADSCs,TGF-?1 is a multifunctional cytokine with anti-inflammatory,reparative,and neuroprotective functions.Whether ADSCs can secrete TGF-?1 and up-regulate the expression of PLOD2,thereby promoting the recovery of spinal cord injury,has become the focus of this study.This study is divided into three parts.The first part:ADSC co-culture promotes survival of mechanically injured neurons.The second part:ADSCs exert neuronal repair functions mainly through the TGF-?1/Smad3 signaling pathway.The third part:ADSCs promote functional recovery after spinal cord injury by activating the TGF-?1/Smad3/PLOD2 signaling pathway.Part ? ADSC co-culture promotes survival of mechanically injured neuronsObjective1.ADSCs were isolated from human adipose tissue,identified according to standard criteria:multipotent differentiation potential and specific surface antigen expression.2.A wound-healing assay involving mechanical injury(MI)was performed on both differentiated PC 12 cells and rat cortical neurons.3.To investigate the influence of ADSCs on neuronal recovery from injury,a neuron-ADSC co-culture system was first established in vitro.Methods1.Culture and Identification of ADSCsADSCs were isolated from human adipose tissue.ADSCs were identified according to standard criteria and used on passages 3-5 for experiments.Differentiation of ADSCs into osteogenic and adipogenic lineages was carried out using a differentiation medium kit.Specific surface antigen expression was evaluated on a flow cytometer.2.Mechanical injury assay and cell co-culturing150ng/mL NGF induced PC 12 cells to differentiate into neural cells,which was identified by MAP2 staining.Take the cerebral cortex of newborn Wistar rats,isolate and culture primary neural cells,and identify them by NeuN staining.PC 12 cells and rat cortical neurons were independently seeded on 6-well plates.Upon confluence,the cultures were scratched at the midline with a 10-?1 pipette tip.3.The influence of ADSCs co-culture on neuronal recovery from MIADSCs were inoculated onto transwells(3×105 cells per well)and co-cultured with injured neurons for 3 days.Following MI,cell proliferation was measured using an EdU staining kit.Cell injury was confirmed by measuring the amount of LDH released.qRT-PCR and Western blot analysis of PLOD2 and neuronal markers MAP2,NSE,GAP43 and GFAP in MI-treated cells co-cultured with ADSCs.Results1.Characterization of ADSCsADSCs in vitro exhibited a fibroblast-like morphology,and differentiated into adipocytes and osteocytes,respectively,after 3 weeks of adipogenic and osteogenic induction.Flow cytometry showed that undifferentiated ADSCs expressed high levels of CD 13,CD44,CD73,CD90 and CD 105,and were negative for the hematopoietic stem cell markers HLA-DR,CD34,and CD45.2.Culture and identification of neuron cellsAfter 5-7 days of differentiation of PC 12 cells induced by 150ng/mL NGF,neurites grew,and the cell morphology was similar to nerve cells,and the result of MAP2 staining was positive.The rat cortical neurons were cultured for 6-8 days in vitro.The cell bodies were small,with axons and dendrites extending from the cell bodies.The axons were slender and NeuN staining was positive.3.ADSCs promote neuronal recovery in vitroResults showed that wound closure rates were significantly higher in cells co-cultured with ADSCs.Moreover,EdU assays showed that co-cultured ADSCs enhanced the proliferation of rat cortical neurons.Accordingly,lactate dehydrogenase(LDH)release assay results indicated that neuronal damage induced by MI was reversed by ADSC co-culturing.qRT-PCR and Western blot results showed that the downregulation of the neuronal markers MAP2,NSE and GAP43 induced by MI was prevented upon ADSC co-culture.Decreased expression of GFAP was also observed in neurons co-cultured with ADSCs.Conclusion1.We successfully isolated mesenchymal stem cells from adipose tissue.2.Our study demonstrated that ADSCs promote the recovery of injured neurons in a co-culture system and reduce neuronal release of LDH.Co-culture of ADSCs can promote the expression of MAP2,NSE and GAP43 in mechanically injured neurons,reduce the expression of GFAP.These results suggest that ADSCs improve neuronal survival,promote axonal regeneration,and reduce glial scar formation after MI.3.Our results demonstrated that co-culture with ADSCs increases the expression of PLOD2 in injured neurons.Part ? ADSCs exert neuronal repair functions mainly through the TGF-?1/Smad3 signaling pathwayObjective1.To investigate whether ADSC-mediated neuronal recovery from MI is mediated by PLOD2 upregulation,a PLOD2 inhibitor,minoxidil,was added into the co-culture system.2.To elucidate the molecular mechanism(s)that mediate ADSC-induced PLOD2 upregulation,we explored potential changes in the expression of TGF-?1 and the effect of exogenous TGF-?1 on gene expression in PC12 cells subjected to MI.3.SB431542 inhibits the TGF-?1/Smad signal pathway,and LY294002 inhibits the PI3K/AKT signal pathway,clarifying the specific signal regulation pathway ofPLOD2.Methods 1.To investigate whether ADSC-mediated neuronal recovery from MI is mediated byPLOD2 upregulation Different concentrations of minoxidil(0.25mM,0.5mM,1.0mM)were applied to PC 12 cells to screen the optimal concentration of minoxidil to inhibit the expression of PLOD2.Immunocytochemistry,EdU and LDH release assay were used to detect the effect of inhibiting PLOD2 expression on the neuroprotection of ADSCs,qRT-PCR and western blot analysis of PLOD2,MAP2,NSE,GAP43,and GFAP expression in MI-treated neurons co-cultured with ADSCs in the presence or absenceof minoxidil.2.The effect of TGF-?1 on neuronal repair functions TGF-?1 levels were measured using a TGF-?1 ELISA Kit.Following MI PC12 cells were treated for 3 days with 0.31,0.62,1.25,2.5,5.0,10,or 20 ng/ml exogenous TGF-?1.qRT-PCR and western blot analysis of PLOD2,P-AKT,AKT,P-Smad3,and Smad2/3 expression in PC12 cells treated with different concentrations of TGF-?1.3.Regulation of PLOD2 expression by TGF-?1/Smad or PI3K/AKT signaling pathwaysSB431542 was added to the ADSCs co-culture system to inhibit the TGF-?1/Smad signaling pathway.We used the PI3K/AKT inhibitor LY294002 to further assess whether PLOD2 expression is influenced by PI3K/AKT signaling in PC12 cells.TGF-?1 levels were measured using a TGF-?1 ELISA Kit.The expression changes of PLOD2,TGF-?1,P-Smad3,Smad2/3,P-AKT,AKT and neural related molecules MAP2,NSE,GAP43,GFAP were detected by qRT-PCR and western blot.Results1.PLOD2 inhibition impairs ADSCs' neuroprotective effectsBased on qRT-PCR and western blot analyses,we selected an experimental concentration of 0.5 mM for minoxidil treatment.MAP2 immunofluorescence showed that the neurorestorative effect of ADSCs was weakened upon inhibition of PLOD2 expression.EdU assay results showed that minoxidil reduced the stimulatory effect of ADSCs on the proliferation of rat cortical neurons,while LDH release assays indicated that ADSC-mediated neuronal protection following MI was attenuated by minoxidil application.Moreover,both qRT-PCR and western blotting confirmed that minoxidil exposure inhibited PLOD2 expression in both PC 12 cells and rat cortical neurons,and attenuated or prevented the changes in MAP2,NSE,and GAP43 expression observed upon ADSC co-culture.2.The effect of TGF-?1 on neuronal repair functionsELISA results showed that TGF-?1 levels were significantly increased in cell culture supernatants from both PC 12 cells and rat cortical neurons upon ADSC co-culture.Results from qRT-PCR assays showed that low TGF-?1 concentrations(0.31-2.5 ng/ml)promoted the expression of PLOD2;this effect was weakened at 5.0 ng/ml,and inhibited instead at TGF-?1 concentrations of 10 and 20 ng/ml.Western blot results showed that low concentrations of TGF-?1 inhibited the expression of P-AKT and promoted the expression of PLOD2,P-Smad3,while high TGF-?1 concentrations had the opposite effect.3.ADSCs exert neuronal repair functions mainly through the TGF-?1/Smad3 signaling pathwayELISA results showed that the expression of TGF-?1 was significantly decreased in the SB/MI/ADSC group.qRT-PCR and western blot analyses showed that the expression of TGF-?1,P-Smad3,and PLOD2 was significantly decreased in the SB/MI/ADSC group.These analyses also showed that the expression of MAP2,NSE,and GAP43 was significantly decreased,while GFAP levels were significantly increased,upon SB/MI/ADSC treatment.qRT-PCR and western blot showed that when the concentration of LY294002 was?20 ?M,the expression of P-AKT and PLOD2 could be significantly inhibited.The above results further confirmed that the AKT signaling pathway can regulate the expression of PLOD2,but when low concentrations of TGF-?1 exist,PLOD2 is mainly regulated by the TGF-?1/Smad signaling pathway.Conclusion1.Inhibited the expression of PLOD2 by minoxidil,the neurorestorative effect of ADSCs was weakened,suggesting that ADSC-mediated neuronal recovery from MI is mediated by PLOD2 upregulation.2.ELISA results showed that TGF-?1 levels were significantly increased in cell culture supernatants from both PC 12 cells and rat cortical neurons upon ADSC co-culture.Low concentration of TGF-?1 inhibits the expression of P-AKT and promotes the expression of P-Smad3 and PLOD2,while high concentration of TGF-?1 is the opposite.3.These observations suggest that ADSCs mediate neuroprotective effects by releasing TGF-?1,which promotes neuronal PLOD2 expression through activation of TGF-?1/Smad3 signaling.Part ? ADSCs promote functional recovery after spinal cord injury by activating the TGF-?1/Smad3/PLOD2 signaling pathway.Objective1.To determine whether a similar neuronal repair functions operates in vivo,a rat SCI model was established.2.The neuroregenerative effect of TGF-?1/Smad3 pathway activation on SCI was confirmed through SB431542 treatment in our rat SCI model in vivo.Methods1.Rat spinal cord injury modelRats were anesthetized by intraperitoneal injection of 10%chloral hydrate,and the T8-T10 lamina was carefully severed to expose the spinal cord.A bulldog clamp was used to hit the spinal cord for 30 s.2.Experimental groupsThree days after injury,SCI rats received PBS,ADSC,and SB431542 treatments.The rats were randomly divided into 5 groups:1)sham;2)PBS;3)ADSC;4)ADSC+SB431542;and 5)SB431542.3.The therapeutic effect of ADSCs transplantation on spinal cord injuryOn days 3,7,14,21,and 28 after drug treatment and/or ADSC transplantation,locomotor recovery was evaluated using the test developed by Basso et al.Rat blood sera were collected three days after ADSC treatment.TGF-?1 levels were measured using a Rat TGF-?1 ELISA Kit.On the 3rd day after drug treatment and/or ADSC transplantation,spinal cord tissues were removed and fixed in 4%paraformaldehyde,processed for H&E,Nissl staining,and immunohistochemical staining.The spinal cord tissue after 3 days of treatment was taken out,and the total protein and total RNA were extracted.qRT-PCR and western blot were used to detect the expression changes of neural cell related genes(MAP2,NSE,GAP43 and GFAP)and PLOD2,TGF-?1,P-Smad3 and Smad2/3.Results1.Transplantation of ADSCs improves functional recovery in experimental models of spinal cord injuryADSCs treatment promoted the recovery of motor function in rats,reduced the lesion volume and the formation of vacuoles,increased the number of Nissl bodies,promoted the expression of MAP2,NSE and GAP43 in spinal cord nerve cells,and inhibit the expression of GFAP.2.TGF-?1 mediates the therapeutic effects of ADSCs in a rat model of SCIADSCs transplantation significantly increased serum levels of TGF-?1 in ADSC-implanted rats.SB431542 transplantation significantly inhibited the recovery of ADSCs on rats' motor function.H&E and Nissl staining showed that SB431542 administration reversed the reduction in lesion volume induced by ADSC transplantation and decreased the formation of Nissl bodies at injured sites.qRT-PCR and western blot assays showed upregulation of TGF-?1,P-Smad3,PLOD2,MAP2,NSE,and GAP43,and downregulation of GFAP expression in SCI samples 3 days after ADSC transplantation.These changes were reversed,however,following SB431542 treatment.Conclusion1.ADSCs promoted functional recovery after their implantation near injury sites in a rat SCI model.2.The neurorestorative effect of ADSCs was weakened after inhibition of PLOD2 expression by SB431542.These findings indicate that ADSCs reduce lesion size and promote functional recovery after SCI mainly through activation of a TGF-?1/P-Samd3/PLOD2 pathway in spinal cord neurons. |
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