Objective:Inflammation in the central nervous system plays a crucial role in the occurrence and development of sepsis-associated encephalopathy.This study aims to explore the effects of maresin 1(MaR1),an anti-inflamm...Objective:Inflammation in the central nervous system plays a crucial role in the occurrence and development of sepsis-associated encephalopathy.This study aims to explore the effects of maresin 1(MaR1),an anti-inflammatory and pro-resolving lipid mediator,on sepsis-induced neuroinflammation and cognitive impairment.Methods:Mice were randomly assigned to 4 groups:A sham group(sham operation+vehicle),a cecal ligation and puncture(CLP)group(CLP operation+vehicle),a MaR1-LD group(CLP operation+1 ng MaR1),and a MaR1-HD group(CLP operation+10 ng MaR1).MaR1 or vehicle was intraperitoneally administered starting 1 h before CLP operation,then every other day for 7 days.Survival rates were monitored,and serum inflammatory cytokines[tumor necrosis factor alpha(TNF-α),interleukin(IL)-1β,and IL-6]were measured 24 h after operation using enzyme-linked immunosorbent assay(ELISA).Cognitive function was assessed 7 days after operation using the Morris water maze(MWM)test and novel object recognition(NOR)task.The mRNA expression of TNF-α,IL-1β,IL-6,inducible nitric oxide synthase(iNOS),IL-4,IL-10,and arginase 1(Arg1)in cortical and hippocampal tissues was determined by real-time reverse transcription PCR(RT-PCR).Western blotting was used to determine the protein expression of iNOS,Arg1,signal transducer and activator of transcription 6(STAT6),peroxisome proliferator-activated receptor gamma(PPARγ),and phosphorylated STAT6(p-STAT6)in hippocampal tissue.Microglia activation was visualized via immunofluorescence.Mice were also treated with the PPARγantagonist GW9662 to confirm the involvement of this pathway in MaR1’s effects.Results:CLP increased serum levels of TNF-α,IL-1β,and IL-6,and reduced body weight and survival rates(all P<0.05).Both 1 ng and 10 ng doses of MaR1 significantly reduced serum TNF-α,IL-1β,and IL-6 levels,improved body weight,and increased survival rates(all P<0.05).No significant difference in efficacy was observed between the 2 doses(all P>0.05).MWM test and NOR task indicated that CLP impaired spatial learning,which MaR1 mitigated.However,GW9662 partially reversed MaR1’s protective effects.Real-time RTPCR results demonstrated that,compared to the sham group,mRNA expression of TNF-α,IL-1β,and iNOS significantly increased in hippocampal tissues following CLP(all P<0.05),while IL-4,IL-10,and Arg1 showed a slight decrease,though the differences were not statistically significant(all P>0.05).Compared to the CLP group,both 1 ng and 10 ng MaR1 decreased TNF-α,IL-1β,and iNOS mRNA expression in hippocampal tissues and increased IL-4,IL-10,and Arg1 mRNA expression(all P<0.05).Immunofluorescence results indicated a significant increase in Iba1-positive microglia in the hippocampus after CLP compared to the sham group(P<0.05).Administration of 1 ng and 10 ng MaR1 reduced the percentage area of Iba1-positive cells in the hippocampus compared to the CLP group(both P<0.05).Western blotting results showed that,compared to the CLP group,both 1 ng and 10 ng MaR1 down-regulated the iNOS expression,while up-regulated the expression of Arg1,PPARγ,and p-STAT6(all P<0.05).However,the inclusion of GW9662 counteracted the MaR1-induced upregulation of Arg1 and PPARγcompared to the MaR1-LD group(all P<0.05).Conclusion:MaR1 inhibits the classical activation of hippocampal microglia,promotes alternative activation,reduces sepsis-induced neuroinflammation,and improves cognitive decline.展开更多
Objective Cerebral palsy(CP)is a prevalent neurodevelopmental disorder acquired during the perinatal period,with periventricular white matter injury(PWMI)serving as its primary pathological hallmark.PWMI is characteri...Objective Cerebral palsy(CP)is a prevalent neurodevelopmental disorder acquired during the perinatal period,with periventricular white matter injury(PWMI)serving as its primary pathological hallmark.PWMI is characterized by the loss of oligodendrocytes(OLs)and the disintegration of myelin sheaths,leading to impaired neural connectivity and motor dysfunction.Neural stem cells(NSCs)represent a promising regenerative source for replenishing lost OLs;however,conventional twodimensional(2D)in vitro culture systems lack the three-dimensional(3D)physiological microenvironment.Microfluidic chip technology has emerged as a powerful tool to overcome this limitation by enabling precise spatial and temporal control over 3D microenvironmental conditions,including the establishment of stable concentration gradients of bioactive molecules.Catalpol,an iridoid glycoside derived from traditional medicinal plants,exhibits dual antioxidant and anti-apoptotic properties.Despite its therapeutic potential,the capacity of catalpol to drive NSC differentiation toward OLs under biomimetic 3D conditions,as well as the underlying molecular mechanisms,remains poorly understood.This study aims to develop a microfluidic-based 3D biomimetic platform to systematically investigate the concentration-dependent effects of catalpol on promoting NSCs-to-OLs differentiation and to elucidate the role of the caveolin-1(Cav-1)signaling pathway in this process.Methods We developed a novel multiplexed microfluidic device featuring parallel microchannels with integrated gradient generators capable of establishing and maintaining precise linear concentration gradients(0-3 g/L catalpol)across 3D NSCs cultures.This platform facilitated the continuous perfusion culture of NSC-derived 3D spheroids,mimicking the dynamic in vivo microenvironment.Real-time cell viability was assessed using Calcein-AM/propidium iodide(PI)dual staining,with fluorescence imaging quantifying live/dead cell ratios.Oligodendrocyte differentiation was evaluated through quantitative reverse transcription polymerase chain reaction(qRT-PCR)for MBP and SOX10 gene expression,complemented by immunofluorescence staining to visualize corresponding protein changes.To dissect the molecular mechanism,the Cav-1-specific pharmacological inhibitor methyl‑β‑cyclodextrin(MCD)was employed to perturb the pathway,and its effects on differentiation markers were analyzed.Results Catalpol demonstrated excellent biocompatibility,with cell viability exceeding 96%across the entire tested concentration range(0-3 g/L),confirming its non-cytotoxic nature.At the optimal concentration of 0-3 g/L,catalpol significantly upregulated both MBP and SOX10 expression(P<0.05,P<0.01),indicating robust promotion of oligodendroglial differentiation.Intriguingly,Cav-1 mRNA expression was progressively downregulated during NSC differentiation into OLs.Further inhibition of Cav-1 with MCD further enhanced this effect,leading to a statistically significant increase in OL-specific gene expression(P<0.05,P<0.01),suggesting Cav-1 acts as a negative regulator of OLs differentiation.Conclusion This study established an integrated microfluidic gradient chip-3D NSC spheroid culture system,which combines the advantages of precise chemical gradient control with physiologically relevant 3D cell culture.The findings demonstrate that 3 g/L catalpol effectively suppresses Cav-1 signaling to drive NSC differentiation into functional OLs.This work not only provides novel insights into the Cav-1-dependent mechanisms of myelination but also delivers a scalable technological platform for future research on remyelination therapies,with potential applications in cerebral palsy and other white matter disorders.The platform’s modular design permits adaptation for screening other neurogenic compounds or investigating additional signaling pathways involved in OLs maturation.展开更多
基金supported by the National Natural Science Foundation (81601728,31500726)the Natural Science Foundation of Hunan Province (2021JJ41002),China。
文摘Objective:Inflammation in the central nervous system plays a crucial role in the occurrence and development of sepsis-associated encephalopathy.This study aims to explore the effects of maresin 1(MaR1),an anti-inflammatory and pro-resolving lipid mediator,on sepsis-induced neuroinflammation and cognitive impairment.Methods:Mice were randomly assigned to 4 groups:A sham group(sham operation+vehicle),a cecal ligation and puncture(CLP)group(CLP operation+vehicle),a MaR1-LD group(CLP operation+1 ng MaR1),and a MaR1-HD group(CLP operation+10 ng MaR1).MaR1 or vehicle was intraperitoneally administered starting 1 h before CLP operation,then every other day for 7 days.Survival rates were monitored,and serum inflammatory cytokines[tumor necrosis factor alpha(TNF-α),interleukin(IL)-1β,and IL-6]were measured 24 h after operation using enzyme-linked immunosorbent assay(ELISA).Cognitive function was assessed 7 days after operation using the Morris water maze(MWM)test and novel object recognition(NOR)task.The mRNA expression of TNF-α,IL-1β,IL-6,inducible nitric oxide synthase(iNOS),IL-4,IL-10,and arginase 1(Arg1)in cortical and hippocampal tissues was determined by real-time reverse transcription PCR(RT-PCR).Western blotting was used to determine the protein expression of iNOS,Arg1,signal transducer and activator of transcription 6(STAT6),peroxisome proliferator-activated receptor gamma(PPARγ),and phosphorylated STAT6(p-STAT6)in hippocampal tissue.Microglia activation was visualized via immunofluorescence.Mice were also treated with the PPARγantagonist GW9662 to confirm the involvement of this pathway in MaR1’s effects.Results:CLP increased serum levels of TNF-α,IL-1β,and IL-6,and reduced body weight and survival rates(all P<0.05).Both 1 ng and 10 ng doses of MaR1 significantly reduced serum TNF-α,IL-1β,and IL-6 levels,improved body weight,and increased survival rates(all P<0.05).No significant difference in efficacy was observed between the 2 doses(all P>0.05).MWM test and NOR task indicated that CLP impaired spatial learning,which MaR1 mitigated.However,GW9662 partially reversed MaR1’s protective effects.Real-time RTPCR results demonstrated that,compared to the sham group,mRNA expression of TNF-α,IL-1β,and iNOS significantly increased in hippocampal tissues following CLP(all P<0.05),while IL-4,IL-10,and Arg1 showed a slight decrease,though the differences were not statistically significant(all P>0.05).Compared to the CLP group,both 1 ng and 10 ng MaR1 decreased TNF-α,IL-1β,and iNOS mRNA expression in hippocampal tissues and increased IL-4,IL-10,and Arg1 mRNA expression(all P<0.05).Immunofluorescence results indicated a significant increase in Iba1-positive microglia in the hippocampus after CLP compared to the sham group(P<0.05).Administration of 1 ng and 10 ng MaR1 reduced the percentage area of Iba1-positive cells in the hippocampus compared to the CLP group(both P<0.05).Western blotting results showed that,compared to the CLP group,both 1 ng and 10 ng MaR1 down-regulated the iNOS expression,while up-regulated the expression of Arg1,PPARγ,and p-STAT6(all P<0.05).However,the inclusion of GW9662 counteracted the MaR1-induced upregulation of Arg1 and PPARγcompared to the MaR1-LD group(all P<0.05).Conclusion:MaR1 inhibits the classical activation of hippocampal microglia,promotes alternative activation,reduces sepsis-induced neuroinflammation,and improves cognitive decline.
基金supported by grants from the Liaoning Province Excellent Talent Program Project(XLYC1902031)Dalian Science and Technology Talent Innovation Plan Grant(2022RG18)Basic Research Project of the Department of Education of Liaoning Province(LJKQZ20222395)。
文摘Objective Cerebral palsy(CP)is a prevalent neurodevelopmental disorder acquired during the perinatal period,with periventricular white matter injury(PWMI)serving as its primary pathological hallmark.PWMI is characterized by the loss of oligodendrocytes(OLs)and the disintegration of myelin sheaths,leading to impaired neural connectivity and motor dysfunction.Neural stem cells(NSCs)represent a promising regenerative source for replenishing lost OLs;however,conventional twodimensional(2D)in vitro culture systems lack the three-dimensional(3D)physiological microenvironment.Microfluidic chip technology has emerged as a powerful tool to overcome this limitation by enabling precise spatial and temporal control over 3D microenvironmental conditions,including the establishment of stable concentration gradients of bioactive molecules.Catalpol,an iridoid glycoside derived from traditional medicinal plants,exhibits dual antioxidant and anti-apoptotic properties.Despite its therapeutic potential,the capacity of catalpol to drive NSC differentiation toward OLs under biomimetic 3D conditions,as well as the underlying molecular mechanisms,remains poorly understood.This study aims to develop a microfluidic-based 3D biomimetic platform to systematically investigate the concentration-dependent effects of catalpol on promoting NSCs-to-OLs differentiation and to elucidate the role of the caveolin-1(Cav-1)signaling pathway in this process.Methods We developed a novel multiplexed microfluidic device featuring parallel microchannels with integrated gradient generators capable of establishing and maintaining precise linear concentration gradients(0-3 g/L catalpol)across 3D NSCs cultures.This platform facilitated the continuous perfusion culture of NSC-derived 3D spheroids,mimicking the dynamic in vivo microenvironment.Real-time cell viability was assessed using Calcein-AM/propidium iodide(PI)dual staining,with fluorescence imaging quantifying live/dead cell ratios.Oligodendrocyte differentiation was evaluated through quantitative reverse transcription polymerase chain reaction(qRT-PCR)for MBP and SOX10 gene expression,complemented by immunofluorescence staining to visualize corresponding protein changes.To dissect the molecular mechanism,the Cav-1-specific pharmacological inhibitor methyl‑β‑cyclodextrin(MCD)was employed to perturb the pathway,and its effects on differentiation markers were analyzed.Results Catalpol demonstrated excellent biocompatibility,with cell viability exceeding 96%across the entire tested concentration range(0-3 g/L),confirming its non-cytotoxic nature.At the optimal concentration of 0-3 g/L,catalpol significantly upregulated both MBP and SOX10 expression(P<0.05,P<0.01),indicating robust promotion of oligodendroglial differentiation.Intriguingly,Cav-1 mRNA expression was progressively downregulated during NSC differentiation into OLs.Further inhibition of Cav-1 with MCD further enhanced this effect,leading to a statistically significant increase in OL-specific gene expression(P<0.05,P<0.01),suggesting Cav-1 acts as a negative regulator of OLs differentiation.Conclusion This study established an integrated microfluidic gradient chip-3D NSC spheroid culture system,which combines the advantages of precise chemical gradient control with physiologically relevant 3D cell culture.The findings demonstrate that 3 g/L catalpol effectively suppresses Cav-1 signaling to drive NSC differentiation into functional OLs.This work not only provides novel insights into the Cav-1-dependent mechanisms of myelination but also delivers a scalable technological platform for future research on remyelination therapies,with potential applications in cerebral palsy and other white matter disorders.The platform’s modular design permits adaptation for screening other neurogenic compounds or investigating additional signaling pathways involved in OLs maturation.
