Objective: The study aimed to explore the protective mechanism of Ganoderic acid A (GAA) in renal fibrosisand to verify that GAA can ameliorate renal fibrosis by regulating the Niemann-pick C1-like 1 (NPC1L1) gene. Meth...Objective: The study aimed to explore the protective mechanism of Ganoderic acid A (GAA) in renal fibrosisand to verify that GAA can ameliorate renal fibrosis by regulating the Niemann-pick C1-like 1 (NPC1L1) gene. Methods:Transforming growth factor beta1 (TGF-β1) was used to treat Human Kidney-2 (HK-2) cells to establish a renal fibrosismodel. The differentially expressed genes in the control (CTRL) group, TGF-β1 group, and TGF-β1 + GAA group werescreened via transcriptome sequencing technology and verified by qPCR and Western blot experiments. The NPC1L1gene overexpression plasmid was constructed. The expression levels of N-cad, E-cad, and Slug-related proteins inCTRL, TGF-β1, TGF-β1+GAA (25 μg/mL), and TGF-β1+GAA (25 μg/mL) + NPC1L1 Overexpression (OE) groupswere detected by qPCR and Western blot analysis. Western blot analysis was used to identify the extracellular matrixassociated proteins Tenascin-C, α-SMA, and fibrosis-related protein Collagen I. Fibrosis marker protein Fibronectinwas detected and quantified by immunofluorescence. Results: Transcriptomic sequencing revealed that TGF-β1stimulation led to 267 differentially regulated genes, with 118 up-regulated and 149 down-regulated, while furthermodulation of 213 genes, comprising 112 up-regulated and 101 down-regulated genes, was observed in the GAAintervention group. The target gene in these processes was found to be NPC1L1 by investigations using GeneOntology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). qPCR and Western blot resultsconfirmed that TGF-β1 increased NPC1L1 expression, which was attenuated by GAA. Additionally, TGF-β1upregulated N-cad and Slug. However, GAA reversed this effect and NPC1L1 overexpression partially rescued theGAA effect. TGF-β1 also decreased E-cad expression, reversed by GAA, and NPC1L1 overexpression antagonized thisreversal. Furthermore, TGF-β1 promoted Collagen I, α-SMA, and Tenascin-C expression, and GAA reduced theselevels, effects that were reversed by NPC1L1 overexpression. Immunofluorescence results showed that TGF-β1increased fibronectin expression, which was decreased by GAA, and increased by NPC1L1 overexpression.Conclusion: GAA ameliorates renal fibrosis by antagonizing NPC1L1 gene expression inhibiting epithelialmesenchymal transition and reducing extracellular matrix formation.展开更多
目的探究干酪乳酪杆菌Zhang(Lacticaseibacillus casei Zhang)在小鼠胆囊胆固醇结石形成过程中对肝脏SR-BI以及小肠NPC1L1、ABCG8基因表达的影响。方法购买8周龄,体重(18.70±0.89)g,SPF级雌性C57BL/6J小鼠40只,适应性喂养1周后,称...目的探究干酪乳酪杆菌Zhang(Lacticaseibacillus casei Zhang)在小鼠胆囊胆固醇结石形成过程中对肝脏SR-BI以及小肠NPC1L1、ABCG8基因表达的影响。方法购买8周龄,体重(18.70±0.89)g,SPF级雌性C57BL/6J小鼠40只,适应性喂养1周后,称重,采用随机数表法分为基础饲料盐水对照组(G-NS组,n=10)、致石饲料盐水对照组(L-NS组,n=10)、基础饲料L.casei Zhang干预组(G-L-casei组,n=10)及致石饲料L.casei Zhang干预组(L-L-casei组,n=10)。给予G-NS组、G-L-casei组小鼠喂饲基础饲料,L-NS组、L-L-casei组小鼠喂饲致石饲料,同时给予G-L-casei组、L-L-casei组小鼠L.casei Zhang菌液灌胃,对照组小鼠灌胃等体积的生理盐水,1次/d,持续8周。饲养8周后,经内眦静脉取血后处死小鼠,检测四组小鼠甘油三酯(TG)、血清总胆固醇(CHO)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)及载脂蛋白(Apo-AI)水平。肉眼与偏光显微镜下观察小鼠胆囊胆固醇结石形成情况,分别采集小鼠肝脏和小肠组织检测SR-BI、NPC1L1、ABCG8表达水平。结果成石率:G-NS组和G-L-casei组小鼠未见胆囊结石,L-NS组、L-L.casei组小鼠均出现胆囊结石,且L-L-casei组成石率较L-NS组低。血清CHO表达水平:L-L-casei组小鼠低于L-NS组,差异有统计学意义(t=3.873,P<0.05)。血清TG水平:L-L-casei组小鼠低于L-NS组,差异有统计学意义(t=3.481,P<0.01)。血清LDL-C水平:L-L-casei组小鼠低于L-NS组(t=3.815,P<0.01)。血清HDL-C水平:L-L-casei组小鼠高于L-NS组(t=3.235,P<0.01),G-L-casei组小鼠高于G-NS组(t=2.649,P<0.05),差异均有统计学意义。血清Apo-AI水平:L-L-casei组小鼠高于L-NS组,差异有统计学意义(t=3.938,P<0.05)。RT-qPCR小肠NPC1L1表达水平:L-L-casei组低于L-NS组,差异有统计学意义(t=3.147,P<0.01),G-NS组低于L-NS组,差异有统计学意义(t=2.537,P<0.05)。RT-qPCR小肠ABCG8表达水平:L-L-casei组高于L-NS组,差异有统计学意义(t=2.829,P<0.05)。RT-qPCR肝脏SR-BI的表达水平:L-L-casei组小鼠高于L-NS组,差异有统计学意义(t=2.446,P<0.05)。结论L.casei Zhang可以有效改善小鼠血清的高脂状态,降低血清CHO、TG、LDL-C水平,升高血清HDL-C和ApoAI水平;同时可以使小肠NPC1L1表达水平降低,小肠ABCG8和肝脏SR-BI表达水平增加,降低小鼠胆囊结石形成概率。展开更多
该研究观察炎症因子IL1-β对高脂负荷下人肾脏系膜细胞(human glomerular mesangial cells, HMCs)C型尼曼匹克蛋白1(Niemann-pick protein c1, NPC1)表达水平的影响,并初步探讨NPC1介导的胆固醇积聚致细胞损伤的作用机制。体外培养HMCs...该研究观察炎症因子IL1-β对高脂负荷下人肾脏系膜细胞(human glomerular mesangial cells, HMCs)C型尼曼匹克蛋白1(Niemann-pick protein c1, NPC1)表达水平的影响,并初步探讨NPC1介导的胆固醇积聚致细胞损伤的作用机制。体外培养HMCs分为正常对照组、高脂组、高脂+炎症组。Western blot测NPC1蛋白含量;酶法测细胞及内质网胆固醇水平;CCK8法测细胞增殖;流式细胞术测细胞周期;荧光定量PCR测NPC1、GRP78、PERK、ATF6、FN、Col IV mRNA;免疫荧光技术测GRP78、FN的表达水平。予U-18666A干预NPC1的功能,观察内质网胆固醇水平、细胞增殖能力、GRP78、FN、Col IV mRNA水平的变化。结果显示,高脂促进NPC1蛋白及mRNA的表达水平,同时细胞总胆固醇及内质网胆固醇浓度增加,增殖加快、S期比例增加、内质网应激相关分子—GRP78、PERK、ATF6 mRNA及系膜基质成分—FN、Col IV mRNA水平均增加, GRP78、FN的荧光强度也增加;炎症进一步上调高脂负荷下上述各指标水平。U-18666A干预后可减轻高脂和炎症导致的内质网胆固醇积聚及细胞损伤。综上,炎症可通过上调NPC1的表达水平,加重高脂负荷下肾系膜细胞及内质网胆固醇积聚,诱发细胞损伤。展开更多
基金sponsored by KeyResearch and Development Project of Science andTechnology Department of Tibet (No. XZ202201ZY0033G).
文摘Objective: The study aimed to explore the protective mechanism of Ganoderic acid A (GAA) in renal fibrosisand to verify that GAA can ameliorate renal fibrosis by regulating the Niemann-pick C1-like 1 (NPC1L1) gene. Methods:Transforming growth factor beta1 (TGF-β1) was used to treat Human Kidney-2 (HK-2) cells to establish a renal fibrosismodel. The differentially expressed genes in the control (CTRL) group, TGF-β1 group, and TGF-β1 + GAA group werescreened via transcriptome sequencing technology and verified by qPCR and Western blot experiments. The NPC1L1gene overexpression plasmid was constructed. The expression levels of N-cad, E-cad, and Slug-related proteins inCTRL, TGF-β1, TGF-β1+GAA (25 μg/mL), and TGF-β1+GAA (25 μg/mL) + NPC1L1 Overexpression (OE) groupswere detected by qPCR and Western blot analysis. Western blot analysis was used to identify the extracellular matrixassociated proteins Tenascin-C, α-SMA, and fibrosis-related protein Collagen I. Fibrosis marker protein Fibronectinwas detected and quantified by immunofluorescence. Results: Transcriptomic sequencing revealed that TGF-β1stimulation led to 267 differentially regulated genes, with 118 up-regulated and 149 down-regulated, while furthermodulation of 213 genes, comprising 112 up-regulated and 101 down-regulated genes, was observed in the GAAintervention group. The target gene in these processes was found to be NPC1L1 by investigations using GeneOntology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). qPCR and Western blot resultsconfirmed that TGF-β1 increased NPC1L1 expression, which was attenuated by GAA. Additionally, TGF-β1upregulated N-cad and Slug. However, GAA reversed this effect and NPC1L1 overexpression partially rescued theGAA effect. TGF-β1 also decreased E-cad expression, reversed by GAA, and NPC1L1 overexpression antagonized thisreversal. Furthermore, TGF-β1 promoted Collagen I, α-SMA, and Tenascin-C expression, and GAA reduced theselevels, effects that were reversed by NPC1L1 overexpression. Immunofluorescence results showed that TGF-β1increased fibronectin expression, which was decreased by GAA, and increased by NPC1L1 overexpression.Conclusion: GAA ameliorates renal fibrosis by antagonizing NPC1L1 gene expression inhibiting epithelialmesenchymal transition and reducing extracellular matrix formation.
文摘腺苷三磷酸结合盒转运蛋白A1(ATP-binding cassette transporter A1,ABCA1)是血浆高密度脂蛋白(high-density lipoprotein,HDL)颗粒形成之初的限速步骤。ABCA1通过膜泡运输脂质至细胞表面的HDL载脂蛋白的作用机制尚未完全阐明。C型尼曼-匹克病(Niemann-Pick disease type C,NPC)主要由NPC1基因突变引起,NPC1蛋白能促进胆固醇和其他脂质从晚期胞内体/溶酶体流入其他细胞结构。ABCA1和NPC1相互作用保持细胞内脂质平衡,与Tangier病和N C P病等病理过程密切相关。
文摘该研究观察炎症因子IL1-β对高脂负荷下人肾脏系膜细胞(human glomerular mesangial cells, HMCs)C型尼曼匹克蛋白1(Niemann-pick protein c1, NPC1)表达水平的影响,并初步探讨NPC1介导的胆固醇积聚致细胞损伤的作用机制。体外培养HMCs分为正常对照组、高脂组、高脂+炎症组。Western blot测NPC1蛋白含量;酶法测细胞及内质网胆固醇水平;CCK8法测细胞增殖;流式细胞术测细胞周期;荧光定量PCR测NPC1、GRP78、PERK、ATF6、FN、Col IV mRNA;免疫荧光技术测GRP78、FN的表达水平。予U-18666A干预NPC1的功能,观察内质网胆固醇水平、细胞增殖能力、GRP78、FN、Col IV mRNA水平的变化。结果显示,高脂促进NPC1蛋白及mRNA的表达水平,同时细胞总胆固醇及内质网胆固醇浓度增加,增殖加快、S期比例增加、内质网应激相关分子—GRP78、PERK、ATF6 mRNA及系膜基质成分—FN、Col IV mRNA水平均增加, GRP78、FN的荧光强度也增加;炎症进一步上调高脂负荷下上述各指标水平。U-18666A干预后可减轻高脂和炎症导致的内质网胆固醇积聚及细胞损伤。综上,炎症可通过上调NPC1的表达水平,加重高脂负荷下肾系膜细胞及内质网胆固醇积聚,诱发细胞损伤。
