BACKGROUND Hepatic stellate cell(HSC)activation is key to liver fibrosis.Targeting DNA methylation shows promise.Zebularine,a methylation inhibitor,may suppress HSC activation via the calcineurin(CaN)/NFAT3 pathway.Ma...BACKGROUND Hepatic stellate cell(HSC)activation is key to liver fibrosis.Targeting DNA methylation shows promise.Zebularine,a methylation inhibitor,may suppress HSC activation via the calcineurin(CaN)/NFAT3 pathway.Magnetic resonance imaging(MRI)is a noninvasive tool for evaluating liver fibrosis evaluation tool,but multiparametric MRI for zebularine’s effects in liver fibrosis mouse models has not been studied.AIM To clarify the anti-fibrosis mechanism and MRI-evaluated efficacy of zebularine.METHODS In vitro,transforming growth factor(TGF)-β1-stimulated human HSCs(LX-2)were treated with zebularine.α-smooth muscle actin,fibrotic and anti-fibrotic gene levels,and regulator of calcineurin1(RCAN1)regulation were measured.In vivo,carbon tetrachloride(CCl_(4))-induced liver fibrosis in mice was treated with zebularine,and fibrosis was evaluated using various biochemical,histopathological,and MRI methods.RESULTS Zebularine upregulated RCAN1.4 protein(P<0.01)and inhibited the CaN/NFAT3 pathway(P<0.05).In HSCs,TGF-β1 reduced anti-fibrotic gene massage RNA(mRNA)and increased fibrotic mRNA(P<0.05),whereas zebularine had the opposite effects(P<0.01,P<0.05).CCl4-treated mice exhibited increases in various fibrosis-related indices,all of which were reversed by zebularine treatment(P<0.05).CONCLUSION Zebularine may reduce LX-2 activation and extracellular matrix deposition via RCAN1.4 and CaN/NFAT3 path-ways.Multiparametric MRI can assess its efficacy,suggesting zebularine’s potential as a liver fibrosis treatment.展开更多
Background: Regulatory B cells(Bregs) is an indispensable element in inducing immune tolerance after liver transplantation. As one of the microRNAs(miRNAs), mi R-29a-3p also inhibits translation by degrading the targe...Background: Regulatory B cells(Bregs) is an indispensable element in inducing immune tolerance after liver transplantation. As one of the microRNAs(miRNAs), mi R-29a-3p also inhibits translation by degrading the target mRNA, and yet the relationship between Bregs and mi R-29a-3p has not yet been fully explored. This study aimed to investigate the impact of miR-29a-3p on the regulation of differentiation and immunosuppressive functions of memory Bregs(m Bregs) and ultimately provide potentially effective therapies in inducing immune tolerance after liver transplantation. Methods: Flow cytometry was employed to determine the levels of Bregs in peripheral blood mononuclear cells. TaqMan low-density array miRNA assays were used to identify the expression of different miRNAs, electroporation transfection was used to induce mi R-29a-3p overexpression and knockdown, and dual luciferase reporter assay was used to verify the target gene of miR-29a-3p. Results: In patients experiencing acute rejection after liver transplantation, the proportions and immunosuppressive function of m Bregs in the circulating blood were significantly impaired. mi R-29a-3p was found to be a regulator of m Bregs differentiation. Inhibition of miR-29a-3p, which targeted nuclear factor of activated T cells 5(NFAT5), resulted in a conspicuous boost in the differentiation and immunosuppressive function of m Bregs. The inhibition of mi R-29a-3p in CD19~+ B cells was capable of raising the expression levels of NFAT5, thereby promoting B cells to differentiate into m Bregs. In addition, the observed enhancement of differentiation and immunosuppressive function of m Bregs upon mi R-29a-3p inhibition was abolished by the knockdown of NFAT5 in B cells. Conclusions: mi R-29a-3p was found to be a crucial regulator for m Bregs differentiation and immunosuppressive function. Silencing mi R-29a-3p could be a potentially effective therapeutic strategy for inducing immune tolerance after liver transplantation.展开更多
BACKGROUND Shikonin is a natural remedy that is effective at treating diabetic wounds.NFAT5 is a potential therapeutic target for diabetes,and mitochondrial function is essen-tial for wound healing.However,the relatio...BACKGROUND Shikonin is a natural remedy that is effective at treating diabetic wounds.NFAT5 is a potential therapeutic target for diabetes,and mitochondrial function is essen-tial for wound healing.However,the relationship among Shikonin,NFAT5,and mitochondrial function has not been thoroughly studied.Here,we offer new per-spectives on the advantages of shikonin for managing diabetes.AIM To assess the therapeutic mechanism of shikonin in diabetic wounds,its rela-tionship with NFAT5,and its protection of mitochondrial function.METHODS Hypertonic cell and diabetic wound mouse models were established.NFAT5 expression was measured through western blotting and immunofluorescence,in vivo and in vitro.Mitochondrial function was evaluated using reactive oxygen species(ROS)detection and JC-1 and Calcein AM dyes.Mitochondrial structures were observed using transmission electron microscopy.The NFAT5/AMPK pathway was analyzed using a transfection vector and an inhibitor.The effect of shikonin on cells under hypertonic conditions via the NFAT5/AMPK pathway was assessed using western blotting.RESULTS Shikonin treatment preserved HaCaT cell viability,while significantly reducing cyclooxygenase-2 expression levels in a high-glucose environment(P<0.05).Additionally,shikonin maintained mitochondrial morphology,enhanced membrane potential,reduced membrane permeability,and decreased ROS levels in HaCaT cells under hyperosmolar stress.Furthermore,shikonin promoted wound healing in diabetic mice(P<0.05).Shikonin also inhibited NFAT5,in vivo and in vitro(P<0.05).Shikonin treatment reduced NFAT5 expression levels,subsequently inhibiting AMPK expression in vitro(P<0.05).Finally,shikonin inhibited several key downstream molecules of the NFAT5/AMPK pathway,including mammalian target of rapamycin,protein kinase B,nuclear factor kappa-light-chain-enhancer of activated B cells,and inducible nitric oxide synthase(P<0.05).CONCLUSION Shikonin protects mitochondria via the NFAT5/AMPK-related pathway and enhances wound healing in diabetes.展开更多
目的:探究游泳和下坡跑通过钙调磷酸酶(CN)/活化T细胞核因子(NFAT)途径对T2DM小鼠骨吸收代谢的影响。方法:采用6周高脂膳食和一次性注射链脲佐菌素(STZ)进行T2DM造模,成功后随机分为T2DM对照组(TC)、T2DM游泳组(TS)和T2DM下坡跑组(TD),...目的:探究游泳和下坡跑通过钙调磷酸酶(CN)/活化T细胞核因子(NFAT)途径对T2DM小鼠骨吸收代谢的影响。方法:采用6周高脂膳食和一次性注射链脲佐菌素(STZ)进行T2DM造模,成功后随机分为T2DM对照组(TC)、T2DM游泳组(TS)和T2DM下坡跑组(TD),另选C57小鼠为正常对照组(ZC)。T2DM小鼠继续高脂膳食,ZC小鼠饲以普通饲料。TS和TD小鼠分别进行8周游泳和下坡跑训练。末次训练24 h后处死小鼠并取材,应用Micro-CT、细胞原代培养、ELISA、RT-PCR及West-blotting等技术方法对骨组织形态计量学指标、OC数量、离子浓度、细胞因子m RNA和蛋白表达等进行检测。结果:TC组股骨中TRAF6、CN、Src-3、PLC、NFATc1、TRAP m RNA及胫骨中Src1和NFATc1蛋白表达上调(P<0.05),血清IP3和Ca2+浓度升高(P<0.05),BMM分化产生的OC总数量和≥10个核OC数量增多(P<0.01)。股骨远端松质骨和皮质骨骨组织形态计量学指标显著下降(P<0.05)。与TC比,TS组股骨中TRAF6、CN、PLC和TRAPm RNA及Src1蛋白表达下调,血清Ca2+浓度下降(P<0.05或P<0.01)。TD组股骨中TRAF6、CN、Src-3、PLC、NFATc1和TRAPm RNA及胫骨中Src1和NFATc1蛋白表达下调,血清IP3和Ca2+浓度下降(P<0.05)。OC总数量和≥10个核OC数量显著减少(P<0.05),松质骨和皮质骨骨组织形态计量学指标显著改善(P<0.05)。与TS比,TD组股骨中TRAF6、Src-3、PLC和TRAP m RNA表达下调及血清IP3和Ca^(2+)浓度下降(P<0.05),OC总数量(P<0.05)下降,松质骨BS/TV增加(P<0.05)。结论:T2DM小鼠骨吸收增强。下坡跑通过抑制T2DM小鼠骨中CN/NFAT途径,减少OC数量,降低骨吸收,改善骨组织形态结构,且其作用效果优于游泳。展开更多
目的:基于活化T细胞核因子2(NFAT2)/环氧化酶-2(COX-2)通路探讨雷公藤多苷片(TWPT)防治糖尿病肾病(DN)肾脏损伤的可能作用机制。方法:选取雄性清洁级SD大鼠42只,适应性喂养1周后随机分为正常组8只,造模组34只。正常组予以正常饲养,造模...目的:基于活化T细胞核因子2(NFAT2)/环氧化酶-2(COX-2)通路探讨雷公藤多苷片(TWPT)防治糖尿病肾病(DN)肾脏损伤的可能作用机制。方法:选取雄性清洁级SD大鼠42只,适应性喂养1周后随机分为正常组8只,造模组34只。正常组予以正常饲养,造模组采用高脂高糖饮食喂养1周后予腹腔注射链脲佐菌素(STZ)法建立DN大鼠模型,除去造模过程中死亡及失败,选取造模成功的24只随机分为模型组、缬沙坦(8.33 mg·kg^(-1)·d^(-1))组、TWPT(5 mg·kg^(-1)·d^(-1))组。正常组和模型组均予等体积生理盐水灌胃,6周后测量体质量,收集大鼠尿液,腹主动脉取血后处死取材,生化检测血清中的尿素氮(BUN)、肌酐(SCr)、丙氨酸氨基转移酶(ALT)、血脂血糖及尿液中的24 h尿蛋白总量(24 h UTP),苏木素-伊红(HE)及马松(Masson)染色观察肾脏病理,酶联免疫吸附测定法(ELISA)检测血清中的NFAT2、COX-2表达水平,蛋白免疫印迹法(Western blot)检测肾组织中NFAT2、COX-2蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测肾组织中NFAT2、COX-2 mRNA表达。结果:与正常组比较,模型组大鼠24 h UTP、BUN、SCr、CHO、TG、FBG及血清NFAT2、COX-2表达显著升高(P<0.01),肾组织中的NFAT2、COX-2蛋白及mRNA表达显著升高(P<0.01),肾脏病理示肾小球体积增大,系膜细胞轻度增生,系膜基质增宽;与模型组比较,TWPT组大鼠24 h UTP、BUN、SCr、CHO、TG、FBG均明显降低(P<0.05,P<0.01);肾脏病理示肾小球形态基本正常,血清中NFAT2、COX-2表达显著降低(P<0.01),肾组织中的NFAT2、COX-2 mRNA和蛋白表达显著下降(P<0.01)。结论:TWPT可减轻DN大鼠24 h UTP、保护肾功能、改善肾脏病理,其作用机制可能与下调血清及肾组织NFAT2/COX-2表达相关。展开更多
基金Supported by the Health Research Foundation of Hunan Provincial Health Commission,No.W20243192Natural Science Foundation of Changsha,No.kq2403086+1 种基金National Natural Science Foundation of China,No.81571784Hunan Provincial Health Commission Hunan Provincial High-level Health Talent Major Scientific Research Project,No.R2023022.
文摘BACKGROUND Hepatic stellate cell(HSC)activation is key to liver fibrosis.Targeting DNA methylation shows promise.Zebularine,a methylation inhibitor,may suppress HSC activation via the calcineurin(CaN)/NFAT3 pathway.Magnetic resonance imaging(MRI)is a noninvasive tool for evaluating liver fibrosis evaluation tool,but multiparametric MRI for zebularine’s effects in liver fibrosis mouse models has not been studied.AIM To clarify the anti-fibrosis mechanism and MRI-evaluated efficacy of zebularine.METHODS In vitro,transforming growth factor(TGF)-β1-stimulated human HSCs(LX-2)were treated with zebularine.α-smooth muscle actin,fibrotic and anti-fibrotic gene levels,and regulator of calcineurin1(RCAN1)regulation were measured.In vivo,carbon tetrachloride(CCl_(4))-induced liver fibrosis in mice was treated with zebularine,and fibrosis was evaluated using various biochemical,histopathological,and MRI methods.RESULTS Zebularine upregulated RCAN1.4 protein(P<0.01)and inhibited the CaN/NFAT3 pathway(P<0.05).In HSCs,TGF-β1 reduced anti-fibrotic gene massage RNA(mRNA)and increased fibrotic mRNA(P<0.05),whereas zebularine had the opposite effects(P<0.01,P<0.05).CCl4-treated mice exhibited increases in various fibrosis-related indices,all of which were reversed by zebularine treatment(P<0.05).CONCLUSION Zebularine may reduce LX-2 activation and extracellular matrix deposition via RCAN1.4 and CaN/NFAT3 path-ways.Multiparametric MRI can assess its efficacy,suggesting zebularine’s potential as a liver fibrosis treatment.
基金supported by grants from the National Natural Science Foundation of China (82070676)Jiangsu Provincial Medi-cal Innovation Center (CXZX202203)Jiangsu Provincial Medi-cal Key Laboratory (ZDXYS202201)。
文摘Background: Regulatory B cells(Bregs) is an indispensable element in inducing immune tolerance after liver transplantation. As one of the microRNAs(miRNAs), mi R-29a-3p also inhibits translation by degrading the target mRNA, and yet the relationship between Bregs and mi R-29a-3p has not yet been fully explored. This study aimed to investigate the impact of miR-29a-3p on the regulation of differentiation and immunosuppressive functions of memory Bregs(m Bregs) and ultimately provide potentially effective therapies in inducing immune tolerance after liver transplantation. Methods: Flow cytometry was employed to determine the levels of Bregs in peripheral blood mononuclear cells. TaqMan low-density array miRNA assays were used to identify the expression of different miRNAs, electroporation transfection was used to induce mi R-29a-3p overexpression and knockdown, and dual luciferase reporter assay was used to verify the target gene of miR-29a-3p. Results: In patients experiencing acute rejection after liver transplantation, the proportions and immunosuppressive function of m Bregs in the circulating blood were significantly impaired. mi R-29a-3p was found to be a regulator of m Bregs differentiation. Inhibition of miR-29a-3p, which targeted nuclear factor of activated T cells 5(NFAT5), resulted in a conspicuous boost in the differentiation and immunosuppressive function of m Bregs. The inhibition of mi R-29a-3p in CD19~+ B cells was capable of raising the expression levels of NFAT5, thereby promoting B cells to differentiate into m Bregs. In addition, the observed enhancement of differentiation and immunosuppressive function of m Bregs upon mi R-29a-3p inhibition was abolished by the knockdown of NFAT5 in B cells. Conclusions: mi R-29a-3p was found to be a crucial regulator for m Bregs differentiation and immunosuppressive function. Silencing mi R-29a-3p could be a potentially effective therapeutic strategy for inducing immune tolerance after liver transplantation.
基金Supported by National Natural Science Foundation of China,No.82104862Zhejiang Provincial Natural Science Foundation of China,No.LTY22E030003Scientific Research Project Foundation of Zhejiang Chinese Medical University,No.2023FSYYZZ01.
文摘BACKGROUND Shikonin is a natural remedy that is effective at treating diabetic wounds.NFAT5 is a potential therapeutic target for diabetes,and mitochondrial function is essen-tial for wound healing.However,the relationship among Shikonin,NFAT5,and mitochondrial function has not been thoroughly studied.Here,we offer new per-spectives on the advantages of shikonin for managing diabetes.AIM To assess the therapeutic mechanism of shikonin in diabetic wounds,its rela-tionship with NFAT5,and its protection of mitochondrial function.METHODS Hypertonic cell and diabetic wound mouse models were established.NFAT5 expression was measured through western blotting and immunofluorescence,in vivo and in vitro.Mitochondrial function was evaluated using reactive oxygen species(ROS)detection and JC-1 and Calcein AM dyes.Mitochondrial structures were observed using transmission electron microscopy.The NFAT5/AMPK pathway was analyzed using a transfection vector and an inhibitor.The effect of shikonin on cells under hypertonic conditions via the NFAT5/AMPK pathway was assessed using western blotting.RESULTS Shikonin treatment preserved HaCaT cell viability,while significantly reducing cyclooxygenase-2 expression levels in a high-glucose environment(P<0.05).Additionally,shikonin maintained mitochondrial morphology,enhanced membrane potential,reduced membrane permeability,and decreased ROS levels in HaCaT cells under hyperosmolar stress.Furthermore,shikonin promoted wound healing in diabetic mice(P<0.05).Shikonin also inhibited NFAT5,in vivo and in vitro(P<0.05).Shikonin treatment reduced NFAT5 expression levels,subsequently inhibiting AMPK expression in vitro(P<0.05).Finally,shikonin inhibited several key downstream molecules of the NFAT5/AMPK pathway,including mammalian target of rapamycin,protein kinase B,nuclear factor kappa-light-chain-enhancer of activated B cells,and inducible nitric oxide synthase(P<0.05).CONCLUSION Shikonin protects mitochondria via the NFAT5/AMPK-related pathway and enhances wound healing in diabetes.
文摘目的:探究游泳和下坡跑通过钙调磷酸酶(CN)/活化T细胞核因子(NFAT)途径对T2DM小鼠骨吸收代谢的影响。方法:采用6周高脂膳食和一次性注射链脲佐菌素(STZ)进行T2DM造模,成功后随机分为T2DM对照组(TC)、T2DM游泳组(TS)和T2DM下坡跑组(TD),另选C57小鼠为正常对照组(ZC)。T2DM小鼠继续高脂膳食,ZC小鼠饲以普通饲料。TS和TD小鼠分别进行8周游泳和下坡跑训练。末次训练24 h后处死小鼠并取材,应用Micro-CT、细胞原代培养、ELISA、RT-PCR及West-blotting等技术方法对骨组织形态计量学指标、OC数量、离子浓度、细胞因子m RNA和蛋白表达等进行检测。结果:TC组股骨中TRAF6、CN、Src-3、PLC、NFATc1、TRAP m RNA及胫骨中Src1和NFATc1蛋白表达上调(P<0.05),血清IP3和Ca2+浓度升高(P<0.05),BMM分化产生的OC总数量和≥10个核OC数量增多(P<0.01)。股骨远端松质骨和皮质骨骨组织形态计量学指标显著下降(P<0.05)。与TC比,TS组股骨中TRAF6、CN、PLC和TRAPm RNA及Src1蛋白表达下调,血清Ca2+浓度下降(P<0.05或P<0.01)。TD组股骨中TRAF6、CN、Src-3、PLC、NFATc1和TRAPm RNA及胫骨中Src1和NFATc1蛋白表达下调,血清IP3和Ca2+浓度下降(P<0.05)。OC总数量和≥10个核OC数量显著减少(P<0.05),松质骨和皮质骨骨组织形态计量学指标显著改善(P<0.05)。与TS比,TD组股骨中TRAF6、Src-3、PLC和TRAP m RNA表达下调及血清IP3和Ca^(2+)浓度下降(P<0.05),OC总数量(P<0.05)下降,松质骨BS/TV增加(P<0.05)。结论:T2DM小鼠骨吸收增强。下坡跑通过抑制T2DM小鼠骨中CN/NFAT途径,减少OC数量,降低骨吸收,改善骨组织形态结构,且其作用效果优于游泳。
文摘目的:基于活化T细胞核因子2(NFAT2)/环氧化酶-2(COX-2)通路探讨雷公藤多苷片(TWPT)防治糖尿病肾病(DN)肾脏损伤的可能作用机制。方法:选取雄性清洁级SD大鼠42只,适应性喂养1周后随机分为正常组8只,造模组34只。正常组予以正常饲养,造模组采用高脂高糖饮食喂养1周后予腹腔注射链脲佐菌素(STZ)法建立DN大鼠模型,除去造模过程中死亡及失败,选取造模成功的24只随机分为模型组、缬沙坦(8.33 mg·kg^(-1)·d^(-1))组、TWPT(5 mg·kg^(-1)·d^(-1))组。正常组和模型组均予等体积生理盐水灌胃,6周后测量体质量,收集大鼠尿液,腹主动脉取血后处死取材,生化检测血清中的尿素氮(BUN)、肌酐(SCr)、丙氨酸氨基转移酶(ALT)、血脂血糖及尿液中的24 h尿蛋白总量(24 h UTP),苏木素-伊红(HE)及马松(Masson)染色观察肾脏病理,酶联免疫吸附测定法(ELISA)检测血清中的NFAT2、COX-2表达水平,蛋白免疫印迹法(Western blot)检测肾组织中NFAT2、COX-2蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测肾组织中NFAT2、COX-2 mRNA表达。结果:与正常组比较,模型组大鼠24 h UTP、BUN、SCr、CHO、TG、FBG及血清NFAT2、COX-2表达显著升高(P<0.01),肾组织中的NFAT2、COX-2蛋白及mRNA表达显著升高(P<0.01),肾脏病理示肾小球体积增大,系膜细胞轻度增生,系膜基质增宽;与模型组比较,TWPT组大鼠24 h UTP、BUN、SCr、CHO、TG、FBG均明显降低(P<0.05,P<0.01);肾脏病理示肾小球形态基本正常,血清中NFAT2、COX-2表达显著降低(P<0.01),肾组织中的NFAT2、COX-2 mRNA和蛋白表达显著下降(P<0.01)。结论:TWPT可减轻DN大鼠24 h UTP、保护肾功能、改善肾脏病理,其作用机制可能与下调血清及肾组织NFAT2/COX-2表达相关。
