通讯作者: 林智峰, 15199382836@163.com

DOI：10.12201/bmr.202504.00063

To explore the optimal intervention time of 1.25 (OH) 2D3 to protect renal interstitialfibrosis based on NF-κB inflammatory pathway

• 摘要：目的:探讨1.25(OH)2D3在NF-κB通路、炎症细胞因子介导的肾间质纤维化中的作用机制及起效时间窗,以往对于1.25(OH)2D3 在肾间质纤维化中的研究多集中于临床实验,对其基于NF-κB通路和炎症细胞因子介导的体外实验研究相对较少。本研究聚焦于此,深入探讨其作用机制及起效时间窗,为肾间质纤维化的研究开辟了新的方向,有助于从细胞和分子水平更全面地理解1.25(OH)2D3 在该疾病中的作用。方法:将TGF-β1诱导的肾间质纤维化模型作为研究对象,将实验分为空白对照组、TGF-β1组、TGF-β1联合24小时、48小时、72小时1.25(OH)2D3干预组,使用倒置相差显微镜观察肾小管上皮细胞形态变化,使用CCK-8试剂盒检测各组细胞不同时间活性变化,免疫荧光明确NF-κBp65于细胞内的位置,western blot明确NF-κBp65、p-NF-κBp65(磷酸化NF-κBp65)、E-cadherin及α-SMA表达水平在不同时间点的变化;使用Elisa检测各组不同时间TNF-α、IL-6的水平,研究整合了倒置相差显微镜、CCK - 8 试剂盒、免疫荧光、western blot和Elisa等多种先进的实验技术手段。这种多技术联合应用的方式能够从细胞形态、活性、蛋白表达及炎症因子水平等多个层面进行全面检测和分析,避免了单一技术的局限性,为深入研究 1.25(OH)2D3对肾间质纤维化的影响提供了丰富而全面的数据支持。结果:相较空白对照组,TGF-β1干预后肾小管上皮细胞活力下降(P＜0.05),NF-κBp65入核增多(P＜0.0001),p-NF-κBp65/NF-κBp65比值升高、E-cadherin表达减少、α-SMA增多(P＜0.05)；TNF-α、IL-6表达量升高(P＜0.05)；用1.25(OH)2D3在干预24小时、48小时、72小时时肾小管上皮细胞活力明显恢复(P＜0.0001)；NF-κBp65入核减少(P＜0.0001)；p-NF-κBp65/NF-κBp65比值下降、E-cadherin表达上升、α-SMA表达减少(P＜0.05)；TNF-α、IL-6表达量下降(P＜0.05),其中24小时时效果最佳。明确1.25(OH)2D3 的作用机制及最佳干预时间窗，可为临床治疗肾间质纤维化提供更精准的用药依据，有助于提高治疗效果，改善患者预后，这在临床实践中具有重要的指导价值，填补了临床对于该药物在肾间质纤维化治疗中精准用药时间窗的认知空白。结论：巧妙整合多种先进实验技术，包括倒置相差显微镜、CCK-8试剂盒、免疫荧光、western blot和Elisa等。通过从细胞形态、活性、蛋白表达及炎症因子水平等多维度进行全面检测与分析，成功获取丰富而全面的数据，有效避免了单一技术的局限性，重点关注1.25（OH）2D3 在NF-κB通路及炎症细胞因子介导的肾间质纤维化中的体外作用机制与起效时间窗。这一独特视角填补了相关领域体外研究的不足，为从细胞和分子层面深入理解该药物在肾间质纤维化进程中的作用开辟了新路径，具有重要的开创性意义。

  关键词： NF-κB信号通路；1.25（OH）2D3；肾间质纤维化；炎症细胞因子; ; ; 

   

  Abstract: 【Abstract】 Objective: To?investigate?the?mechanism?and?onset?time?window?of?1.25(OH)?D?in?renal?interstitial?fibrosis?mediated?by?the?NF?-?κB?pathway?and?inflammatory?cytokines.?Previous?studies?on?1.25(OH)?D??in?renal?interstitial?fibrosis?mainly?focused?on?clinical?trials,?and?relatively?few?in?vitro?experimental?studies?based?on?the?NF?-?κB?pathway?and?inflammatory?cytokine?mediation.This?study?focuses?on?this?area?to?deeply?explore?its?mechanism?and?onset?time?window,?opening?up?a?new?direction?for?the?research?of?renal?interstitial?fibrosis?and?helping?to?more?comprehensively?understand?the?role?of?1.25(OH)?D??in?this?disease?at?the?cellular?and?molecular?levels.Methods: The?renal?interstitial?fibrosis?model?induced?by?TGF?-?β1?was?used?as?the?research?object.?The?experiment?was?divided?into?a?blank?control?group,?a?TGF?-?β1?group,?and?TGF?-?β1?combined?with?1.25(OH)?D??intervention?groups?for?24?hours,?48?hours,?and?72?hours.?An?inverted?phase?contrast?microscope?was?used?to?observe?the?morphological?changes?of?renal?tubular?epithelial?cells,?the?CCK?-?8?kit?was?used?to?detect?the?activity?changes?of?cells?in?each?group?at?different?times,?immunofluorescence?was?used?to?determine?the?location?of?NF?-?κBp65?in?cells,?western?blot?was?used?to?clarify?the?expression?levels?of?NF?-?κBp65,?p?-?NF?-?κBp65?(phosphorylated?NF?-?κBp65),?E?-?cadherin,?and?α?-?SMA?at?different?time?points,?and?Elisa?was?used?to?detect?the?levels?of?TNF?-?α?and?IL?-?6?in?each?group?at?different?times.?This?study?integrated?multiple?advanced?experimental?techniques?such?as?inverted?phase?contrast?microscope,?CCK?-?8?kit,?immunofluorescence,?western?blot,?and?Elisa.?This?multi?-?technique?combined?application?can?conduct?comprehensive?detection?and?analysis?from?multiple?aspects?such?as?cell?morphology,?activity,?protein?expression,?and?inflammatory?factor?levels,?avoiding?the?limitations?of?a?single?technique?and?providing?rich?and?comprehensive?data?support?for?in?-?depth?research?on?the?impact?of?1.25(OH)?D??on?renal?interstitial?fibrosis. Results: Compared with the blank control group, after TGF - β1 intervention, the viability of renal tubular epithelial cells decreased (P<0.05), the nuclear translocation of NF-κBp65 increased (P<0.0001), the ratio of p-NF-κBp65/NF-κBp65 increased, the expression of E-cadherin decreased, and α-SMA increased (P<0.05); the expression levels of TNF-α and IL-6 increased (P <0.05). When treated with 1.25(OH)?D? for 24 hours, 48 hours, and 72 hours, the viability of renal tubular epithelial cells was significantly restored (P<0.0001); the nuclear translocation of NF - κBp65 decreased (P<0.0001); the ratio of p - NF-κBp65/NF-κBp65 decreased, the expression of E-cadherin increased, and the expression of α-SMA decreased (P<0.05); the expression levels of TNF-α and IL-6 decreased (P<0.05), and the effect was the best at 24 hours. Clarifying the mechanism and optimal intervention time window of 1.25(OH)?D? can provide a more accurate basis for clinical treatment of renal interstitial fibrosis, help improve the treatment effect, and improve the prognosis of patients, which has important guiding value in clinical practice and fills the cognitive gap in the precise medication time window of this drug in the treatment of renal interstitial fibrosis in the clinic. Conclusion: This study skillfully integrated multiple advanced experimental techniques, including inverted phase contrast microscope, CCK-8 kit, immunofluorescence, western blot, and Elisa. Through comprehensive detection and analysis from multiple dimensions such as cell morphology, activity, protein expression, and inflammatory factor levels, rich and comprehensive data were successfully obtained, effectively avoiding the limitations of a single technique. It focused on the in vitro mechanism and onset time window of 1.25(OH)?D? in renal interstitial fibrosis mediated by the NF-κB pathway and inflammatory cytokines. This unique perspective fills the gap in in vitro research in related fields and opens up a new path for in - depth understanding of the role of this drug in the process of renal interstitial fibrosis at the cellular and molecular levels, which is of great pioneering significance.

  Key words: NF-κB signaling pathway; 1,25(OH)2D3; Renal interstitial fibrosis; Inflammatory cytokines

  提交时间：2025-04-20

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