Autophagy-induced mesenchymal stem cell-derived extracellular vesicles ameliorated renal fibrosis in an in vitro model.

IF 2.2 4区工程技术 Q3 PHARMACOLOGY & PHARMACY Bioimpacts Pub Date : 2023-01-01 Epub Date: 2022-08-13 DOI:10.34172/bi.2022.24256

Behnaz Ahrabi, Hojjat Allah Abbaszadeh, Abbas Piryaei, Faezeh Shekari, Navid Ahmady Roozbahany, Mahya Rouhollahi, Forough Azam Sayahpour, Mahnaz Ahrabi, Hadi Azimi, Reza Moghadasali

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Abstract

Introduction: Chronic and progressive damage to the kidney by inflammatory processes, may lead to an increase in the extracellular matrix production, a condition known as renal fibrosis. The current study aims to evaluate if the extracellular vesicles (EVs) derived from autophagic adipose-derived mesenchymal stem cells (ADMSCs) can reduce the inflammation and extracellular matrix accumulation in damaged kidney tissue.

Methods: Autophagy was induced in ADMSCs using 2µM concentration curcumin and was confirmed by evaluating LC3B, ATG7, and Beclin1 using real-time polymerase chain reaction (PCR) and Western blot. An in vitro renal fibrotic model was established in HEK-293 cells exposed to H2O2 (0.8mM) for 24 and 72 hours. The fibrotic model was confirmed through evaluation of collagen I, transforming growth factor-beta 1 (TGF-β1), E-cadherin, and vimentin genes expression using real-time PCR, collagen I protein by ELISA. After induction of fibrosis for 24 and 72 hours, the HEK cells were treated with NEVs (non-autophagy EVs) (50µM) or AEVs (autophagy EVs) (50µM) at 48, 96, and 124 hours, and then the samples were collected at 72 and 148 hours. Expression of collagen I, TGF-β1, E-cadherin, and vimentin Genes was evaluated via RT-PCR, and protein levels of IL1, TNF-α, IL4, IL10 using ELISA.

Results: Induction of autophagy using curcumin (2µM) for 24 hours significantly increased LC3B, Beclin1, and ATG7 in the ADMSCs. Upregulation in anti-fibrotic (E-cadherin) and anti-inflammatory (IL4, IL10) gene expression was significantly different in the fibrotic model treated by AEVs compared to NEVs. Also, the downregulation of fibrotic (TGF-β1, vimentin, collagen I) and pro-inflammatory (IL1, TNFα) gene expression was significantly different in AEVs compared with those treated by NEVs.

Conclusion: Our findings suggest that AEVs can be considered as a therapeutic modality for renal fibrosis in the future.

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自噬诱导的间充质干细胞衍生的细胞外小泡在体外模型中改善肾纤维化。

引言：炎症过程对肾脏的慢性和进行性损伤，可能导致细胞外基质产生增加，这种情况被称为肾纤维化。目前的研究旨在评估来自自噬脂肪来源的间充质干细胞（ADMSC）的细胞外小泡（EVs）是否可以减少受损肾组织中的炎症和细胞外基质积聚。方法：使用2µM浓度的姜黄素在ADMSC中诱导自噬，并通过使用实时聚合酶链式反应（PCR）和蛋白质印迹评估LC3B、ATG7和Beclin1来证实。在暴露于H2O2（0.8mM）24和72小时的HEK-293细胞中建立体外肾纤维化模型。通过实时PCR评估I型胶原、转化生长因子β1（TGF-β1）、E-钙粘蛋白和波形蛋白基因表达，通过ELISA评估I型胶原蛋白，证实了纤维化模型。在诱导纤维化24和72小时后，在48、96和124小时用NEV（非自噬EVs）（50µM）或AEV（自噬EVs）（50μM）处理HEK细胞，然后在72和148小时收集样品。通过RT-PCR评估I型胶原、TGF-β1、E-钙粘蛋白和波形蛋白基因的表达，并使用ELISA评估IL1、TNF-α、IL4和IL10的蛋白水平。结果：使用姜黄素（2µM）诱导自噬24小时可显著增加ADMSC中的LC3B、Beclin1和ATG7。在AEVs治疗的纤维化模型中，抗纤维化（E-钙粘蛋白）和抗炎（IL4，IL10）基因表达的上调与NEVs相比有显著差异。此外，与NEVs治疗的患者相比，AEVs对纤维化（TGF-β1、波形蛋白、I型胶原）和促炎（IL1、TNFα）基因表达的下调也有显著差异。

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来源期刊

Bioimpacts Pharmacology, Toxicology and Pharmaceutics-Pharmaceutical Science

CiteScore

4.80

自引率

7.70%

发文量

审稿时长

5 weeks

期刊介绍： BioImpacts (BI) is a peer-reviewed multidisciplinary international journal, covering original research articles, reviews, commentaries, hypotheses, methodologies, and visions/reflections dealing with all aspects of biological and biomedical researches at molecular, cellular, functional and translational dimensions.