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

Corresponding author: Lin ZhiFeng, 15199382836@163.com

DOI: 10.12201/bmr.202504.00063

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  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

  Submit time: 20 April 2025

  Copyright: The copyright holder for this preprint is the author/funder, who has granted biomedRxiv a license to display the preprint in perpetuity.
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