Adipose mesenchymal stem cell-derived extracellular vesicles containing microRNA-26a-5p target TLR4 and protect against diabetic nephropathy
2020; Elsevier BV; Volume: 295; Issue: 37 Linguagem: Inglês
10.1074/jbc.ra120.012522
ISSN1083-351X
AutoresYurui Duan, Qingyang Luo, Yun Wang, Yali Ma, Fang Chen, Xiaoguang Zhu, Jun Shi,
Tópico(s)Circular RNAs in diseases
ResumoDiabetic nephropathy (DN) is a complication of diabetes that is increasing in prevalence in China. Extracellular vesicles (EVs) carrying microRNAs (miRs) may represent a useful tool in the development of therapies for DN. Here, we report that EVs released by adipose-derived mesenchymal stem cells (ADSCs) during DN contain a microRNA, miR-26a-5p, that suppresses DN. Using bioinformatic analyses, we identified differentially expressed miRs in EVs from ADSCs and in DN and predicted downstream regulatory target genes. We isolated mesenchymal stem cells (MSCs) from adipose tissues and collected EVs from the ADSCs. We exposed mouse glomerular podocytes and MP5 cells to high glucose (HG), ADSC-derived EVs, miR-26a-5p inhibitor/antagomir, Toll-like receptor 4 (TLR4) plasmids, or the NF-κB pathway activator (phorbol-12-myristate-13-acetate, or PMA). We used the cell counting kit-8 (CCK-8) assay and flow cytometry to investigate the impact of miR-26a-5p on cell viability and apoptosis and validated the results of these assays with in vivo experiments in nude mice. We found that in DN, miR-26a-5p is expressed at very low levels, whereas TLR4 is highly expressed. Of note, EVs from ADSCs ameliorated the pathological symptoms of DN in diabetic mice and transferred miR-26a-5p to HG-induced MP5 cells, improving viability while suppressing the apoptosis of MP5 cells. We also found that miR-26a-5p protects HG-induced MP5 cells from injury by targeting TLR4, inactivating the NF-κB pathway, and downregulating vascular endothelial growth factor A (VEGFA). Moreover, ADSC-derived EVs transferred miR-26a-5p to mouse glomerular podocytes, which ameliorated DN pathology. These findings suggest that miR-26a-5p from ADSC-derived EVs protects against DN. Diabetic nephropathy (DN) is a complication of diabetes that is increasing in prevalence in China. Extracellular vesicles (EVs) carrying microRNAs (miRs) may represent a useful tool in the development of therapies for DN. Here, we report that EVs released by adipose-derived mesenchymal stem cells (ADSCs) during DN contain a microRNA, miR-26a-5p, that suppresses DN. Using bioinformatic analyses, we identified differentially expressed miRs in EVs from ADSCs and in DN and predicted downstream regulatory target genes. We isolated mesenchymal stem cells (MSCs) from adipose tissues and collected EVs from the ADSCs. We exposed mouse glomerular podocytes and MP5 cells to high glucose (HG), ADSC-derived EVs, miR-26a-5p inhibitor/antagomir, Toll-like receptor 4 (TLR4) plasmids, or the NF-κB pathway activator (phorbol-12-myristate-13-acetate, or PMA). We used the cell counting kit-8 (CCK-8) assay and flow cytometry to investigate the impact of miR-26a-5p on cell viability and apoptosis and validated the results of these assays with in vivo experiments in nude mice. We found that in DN, miR-26a-5p is expressed at very low levels, whereas TLR4 is highly expressed. Of note, EVs from ADSCs ameliorated the pathological symptoms of DN in diabetic mice and transferred miR-26a-5p to HG-induced MP5 cells, improving viability while suppressing the apoptosis of MP5 cells. We also found that miR-26a-5p protects HG-induced MP5 cells from injury by targeting TLR4, inactivating the NF-κB pathway, and downregulating vascular endothelial growth factor A (VEGFA). Moreover, ADSC-derived EVs transferred miR-26a-5p to mouse glomerular podocytes, which ameliorated DN pathology. These findings suggest that miR-26a-5p from ADSC-derived EVs protects against DN. Diabetic nephropathy (DN), a common microvascular complication of diabetes, is one of the major causes of death among patients with diabetes (1Wang X. Xu Y. Chu C. Li H. Mi J. Wen Z. Zhang S. Wang Q. Quan S. Effect of safflower yellow on early type II diabetic nephropathy: a systematic review and meta-analysis of randomized controlled trials.J. Pediatr. Endocrinol. Metab. 2019; 32: 653-66510.1515/jpem-2018-0425Crossref PubMed Scopus (6) Google Scholar). It is also the leading cause of both chronic kidney and end-stage renal diseases worldwide (2De la Cruz-Cano E. Jimenez-Gonzalez C.D.C. Morales-Garcia V. Pineda-Perez C. Tejas-Juarez J.G. Rendon-Gandarilla F.J. Jimenez-Morales S. Diaz-Gandarilla J.A. Arg913Gln variation of SLC12A3 gene is associated with diabetic nephropathy in type 2 diabetes and Gitelman syndrome: a systematic review.BMC Nephrol. 2019; 20 (31660880): 39310.1186/s12882-019-1590-9Crossref PubMed Scopus (9) Google Scholar). 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Glomerular mesangial cell and podocyte injuries in diabetic nephropathy.Nephrology. 2018; 23 (30298646): 32-3710.1111/nep.13451Crossref PubMed Scopus (102) Google Scholar), and DN progression can be slowed by reducing podocyte injury (7Zhong F. Chen H. Xie Y. Azeloglu E.U. Wei C. Zhang W. Li Z. Chuang P.Y. Jim B. Li H. Elmastour F. Riyad J.M. Weber T. Chen H. Wang Y. et al.Protein S protects against podocyte injury in diabetic nephropathy.J. Am. Soc. Nephrol. 2018; 29 (29511111): 1397-141010.1681/ASN.2017030234Crossref PubMed Scopus (29) Google Scholar). Current treatment methods for DN mainly rely on the management of hyperglycemia and blood pressure (8Wang Y. Wang C. Zhang X. Gu H.F. Wu L. Common drugs for stabilization of renal function in the progression of diabetic nephropathy and their relations with hypertension therapy.Curr. Diabetes Rev. 2018; 14 (28201968): 149-16110.2174/1573399813666170214112115Crossref PubMed Scopus (7) Google Scholar). Evidence suggests that extracellular vesicles (EVs) slow the development of DN by protecting podocytes from injury (9Duan Y.R. Chen B.P. Chen F. Yang S.X. Zhu C.Y. Ma Y.L. Li Y. Shi J. Exosomal microRNA-16-5p from human urine-derived stem cells ameliorates diabetic nephropathy through protection of podocyte.J. Cell Mol. Med. 2019; 10.1111/jcmm.14558Crossref Scopus (45) Google Scholar). Elucidating mechanisms by which EVs regulate podocyte injury may represent a therapeutic target for the treatment of DN. EVs are nano-sized membrane vesicles that include exosomes and microvesicles, and they are released by many cells types, including mesenchymal stem cells (MSCs) (10Gangadaran P. Ahn B.C. In vivo tracking of tumor-derived bioluminescent extracellular vesicles in mice.Methods Mol. Biol. 2020; 2081 (31721127): 203-21010.1007/978-1-4939-9940-8_14Crossref PubMed Scopus (4) Google Scholar, 11Fiedler T. Rabe M. Mundkowski R.G. Oehmcke-Hecht S. Peters K. Adipose-derived mesenchymal stem cells release microvesicles with procoagulant activity.Int. J. Biochem. Cell Biol. 2018; 100 (29778527): 49-5310.1016/j.biocel.2018.05.008Crossref PubMed Scopus (14) Google Scholar). MSCs are multipotent stem cells that differentiate into a variety of lineages and exert important functions in bone regeneration and repair (12Yang Q. Jia L. Li X. Guo R. Huang Y. Zheng Y. Li W. Long noncoding RNAs: new players in the osteogenic differentiation of bone marrow- and adipose-derived mesenchymal stem cells.Stem Cell Rev. Rep. 2018; 14 (29464508): 297-30810.1007/s12015-018-9801-5Crossref PubMed Scopus (45) Google Scholar). A recent study highlighted the role of exosomes derived from adipose-derived mesenchymal stem cells (ADSCs) in protecting against the development of DN (13Jin J. Shi Y. Gong J. Zhao L. Li Y. He Q. Huang H. Exosome secreted from adipose-derived stem cells attenuates diabetic nephropathy by promoting autophagy flux and inhibiting apoptosis in podocyte.Stem Cell Res. Ther. 2019; 10 (30876481): 9510.1186/s13287-019-1177-1Crossref PubMed Scopus (140) Google Scholar). EVs are known to contain mRNA, microRNAs (miRs), and long noncoding RNAs, thereby transferring genetic information from one cell to the next (14Tetta C. Ghigo E. Silengo L. Deregibus M.C. Camussi G. Extracellular vesicles as an emerging mechanism of cell-to-cell communication.Endocrine. 2013; 44 (23203002): 11-1910.1007/s12020-012-9839-0Crossref PubMed Scopus (258) Google Scholar). Interestingly, miR-26a-5p serves as a candidate biomarker for DN by a meta-analysis of profiling studies (15Gholaminejad A. Abdul Tehrani H. Gholami Fesharaki M. Identification of candidate microRNA biomarkers in diabetic nephropathy: a meta-analysis of profiling studies.J. Nephrol. 2018; 31 (30019103): 813-83110.1007/s40620-018-0511-5Crossref PubMed Scopus (45) Google Scholar). Our results also showed that miR-26a-5p is highly expressed in EVs derived from MSCs. Loss of miR-26a led to the development of DN in both cultured podocytes and streptozotocin-induced diabetic mice (16Koga K. Yokoi H. Mori K. Kasahara M. Kuwabara T. Imamaki H. Ishii A. Mori K.P. Kato Y. Ohno S. Toda N. Saleem M.A. Sugawara A. Nakao K. Yanagita M. et al.MicroRNA-26a inhibits TGF-beta-induced extracellular matrix protein expression in podocytes by targeting CTGF and is downregulated in diabetic nephropathy.Diabetologia. 2015; 58 (26063197): 2169-218010.1007/s00125-015-3642-4Crossref PubMed Scopus (84) Google Scholar). Additionally, miR-26a-5p was suggested to be one of the therapeutic mediators in DN (15Gholaminejad A. Abdul Tehrani H. Gholami Fesharaki M. Identification of candidate microRNA biomarkers in diabetic nephropathy: a meta-analysis of profiling studies.J. Nephrol. 2018; 31 (30019103): 813-83110.1007/s40620-018-0511-5Crossref PubMed Scopus (45) Google Scholar). Based on these findings, we hypothesized that EV-derived miR-26a-5p could regulate DN. Toll-like receptor 4 (TLR4), which serves as a signaling receptor for lipopolysaccharides and as a crucial regulator of the innate immunity system, was identified as a downstream target gene of miR-26a-5p (17Kolz M. Baumert J. Muller M. Khuseyinova N. Klopp N. Thorand B. Meisinger C. Herder C. Koenig W. Illig T. Association between variations in the TLR4 gene and incident type 2 diabetes is modified by the ratio of total cholesterol to HDL-cholesterol.BMC Med. Genet. 2008; 9: 910.1186/1471-2350-9-9Crossref PubMed Scopus (30) Google Scholar). One study used M4200 cells to show that TLR4 also participates in the pathogenesis of DN (18Liu Z.M. Zheng H.Y. Chen L.H. Li Y.L. Wang Q. Liao C.F. Li X.W. Low expression of miR-203 promoted diabetic nephropathy via increasing TLR4.Eur. Rev. Med. Pharmacol. Sci. 2018; 22 (30229838): 5627-563410.26355/eurrev_201809_15828PubMed Google Scholar). Moreover, downregulation of TLR/NF-κB (NF-κB) using umbelliferone ameliorated renal function in a rat model of DN (19Wang H.Q. Wang S.S. Chiufai K. Wang Q. Cheng X.L. Umbelliferone ameliorates renal function in diabetic nephropathy rats through regulating inflammation and TLR/NF-kappaB pathway.Chin. J. Nat. Med. 2019; 17 (31171269): 346-35410.1016/S1875-5364(19)30040-8PubMed Google Scholar). Notably, the activation of NF-κB is thought to be related to the inflammation and disease development that are associated with DN (20Li M. Guo Q. Cai H. Wang H. Ma Z. Zhang X. miR-218 regulates diabetic nephropathy via targeting IKK-beta and modulating NK-kappaB-mediated inflammation.J. Cell. Physiol. 2020; 235: 3362-337110.1002/jcp.29224Crossref PubMed Scopus (32) Google Scholar). Upregulation of NF-κB by TLR4 was previously reported in rat pineal glands and human umbilical vein endothelial cells (21da Silveira Cruz-Machado S. Carvalho-Sousa C.E. Tamura E.K. Pinato L. Cecon E. Fernandes P.A. de Avellar M.C. Ferreira Z.S. Markus R.P. TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway.J. Pineal Res. 2010; 49 (20586888): 183-19210.1111/j.1600-079X.2010.00785.xPubMed Google Scholar, 22Ni H. Zhao W. Kong X. Li H. Ouyang J. Celastrol inhibits lipopolysaccharide-induced angiogenesis by suppressing TLR4-triggered nuclear factor-kappa B activation.Acta Haematol. 2014; 131 (24157922): 102-11110.1159/000354770Crossref PubMed Scopus (40) Google Scholar). In addition, the NF-κB pathway could promote the expression of vascular endothelial growth factor A (VEGFA) (23Liang S. Chen Z. Jiang G. Zhou Y. Liu Q. Su Q. Wei W. Du J. Wang H. Activation of GPER suppresses migration and angiogenesis of triple negative breast cancer via inhibition of NF-kappaB/IL-6 signals.Cancer Lett. 2017; 386 (27836733): 12-2310.1016/j.canlet.2016.11.003Crossref PubMed Scopus (83) Google Scholar), which contributes to glomerular endothelial cell dysfunction as well as albuminuria in DN (24Onions K.L. Gamez M. Buckner N.R. Baker S.L. Betteridge K.B. Desideri S. Dallyn B.P. Ramnath R.D. Neal C.R. Farmer L.K. Mathieson P.W. Gnudi L. Alitalo K. Bates D.O. Salmon A.H.J. et al.VEGFC reduces glomerular albumin permeability and protects against alterations in VEGF receptor expression in diabetic nephropathy.Diabetes. 2019; 68 (30389746): 172-18710.2337/db18-0045Crossref PubMed Scopus (32) Google Scholar). Taking these findings into consideration, we hypothesized that ADSC-derived EVs transferred miR-26a-5p to other cells regulating the progression of DN by targeting TLR4 and modulating NF-κB and VEGFA. Thus, this study may help to identify a novel strategy for the control of DN. Analysis of the EVmiRNA database revealed that the expression of miR-26a-5p in the EVs derived from MSCs was significantly upregulated (Fig. 1A). Furthermore, the miRanda database and Starbase database were used to predict the downstream target genes of differentially expressed miRNAs. These results were then compared with a diabetes-related gene expression data set (GSE21340). Five genes were identified at the intersection of these analyses (SMAD1, CELF2, SMAD6, PYGL, and TLR4) (Fig. 1B). Because the miRanda database predicted a binding site between miR-26a-5p and TLR4 (Fig. 1C), we chose to investigate the expression of TLR4 in the GSE21340 data set. We found that TLR4 expression was significantly higher in DN (Fig. 1D). These results suggest that miR-26a-5p delivered by ADSC-derived EVs protect against DN by regulating TLR4. ADSCs were isolated from the subcutaneous adipose tissues of C57BL/KsJ db/m mice. The surface markers associated with ADSCs were detected by flow cytometry after the third passage. ADSCs were positive for CD29 (96.7%), CD44 (95.2%), CD73 (99.1%), CD90 (98.4%), and HLA-A, -B, and -C (98.5%) but negative for CD14 (4.2%), CD19 (0.26%), CD34 (1.9%), CD45 (1.2%), and HLA-DR (0.89%) (Fig. 2A). The pluripotency of isolated ADSCs was tested. After isolation and culture, ADSCs could be differentiated into osteogenic, lipogenic, and chondrogenic cell types (Fig. 2B). The above results demonstrated that ADSCs isolated from adipose tissues had the property of pluripotent stem cell differentiation. Next, EVs isolated from medium used to grow ADSCs were characterized. Dynamic light scattering (DLS) analysis indicated that EVs range in diameter from 30–150 nm. Transmission EM revealed the morphology of EVs to be cup-shaped or spherical. In addition, Western blot analysis of marker proteins of EVs confirmed the expression of CD63 and TSG101, whereas calnexin was absent, indicating that EVs had been successfully isolated (Fig. 2, C–E). The above results demonstrated the successful isolation of EVs from ADSCs. To evaluate the mouse model of spontaneous diabetes, we compared the levels of protein found in the urine (within 24 h), serum creatinine (Scr), and blood urea nitrogen (BUN) in C57BL/KsJ db/db spontaneous diabetic mice and C57BL/KsJ db/m control mice. The spontaneously diabetic mice had twice the amount of urine protein and 3 times as much Scr and BUN as the control mice (Fig. 3, A–C). The histopathological phenotype was characterized by periodic acid Schiff (PAS) staining of DN tissues. Spontaneously diabetic mice showed an accumulation of extracellular matrix in their kidney tissues, viability of mesangial cells, and a thickening of basement membrane. In addition, there was increased proteinuria in the lumen, hyaline degeneration, and severe vacuolar degeneration of renal tubular epithelial cells in these mice compared with that of control mice, indicating the successful establishment of the DN cell model (Fig. 3D). To investigate the effect of EVs from ADSCs on spontaneous diabetic mice, we injected ADSC-derived EVs or PBS into mice via the tail vein, and then changes of urine protein, Scr, and BUN levels were subsequently measured, followed by PAS staining to detect histopathological changes. The results illustrated that treatment with EVs derived from ADSCs notably reduced the levels of urine protein, Scr, and BUN in the DN mouse model and alleviated the histopathological changes associated with DN (Fig. 3, A–D). Next, TUNEL staining was used to detect the apoptosis of glomerular podocytes in mice. Glomerular podocytes from spontaneously diabetic mice (C57BL/KsJ db/db) exhibited a significant increase in apoptosis compared with control mice (C57BL/KsJ db/m mice), and this was reduced by injection of EVs from ADSCs (Fig. 3E). In addition, apoptosis-related proteins (caspase-3, cleaved caspase-3, Bcl-2, Bax) from diabetic mice (C57BL/KsJ db/db) and control (C57BL/KsJ db/m) mice were compared. Bcl-2 protein was significantly reduced in diabetic mice compared with control mice, whereas the protein expression of Bax, cleaved caspase-3, and caspase-3 was increased. Injection of EVs from ADSCs reversed these trends (Fig. 3F). These results demonstrated that ADSC-derived EVs are capable of alleviating the pathological symptoms and inhibiting glomerular podocyte apoptosis in DN mice. The ability of mouse glomerular podocytes, MP5 cells, to internalize EVs from ADSCs was assessed using green fluorescent (PKH67)-labeled EVs. The presence of green fluorescence in MP5 cells was observed by fluorescence microscopy, which indicated that MP5 cells were able to internalize ADSC-derived EVs (Fig. 4A). To simulate the DN cell model in vitro, MP5 cells were induced by HG and treated with ADSC-derived EVs (25 μg/ml). Cell viability was detected by CCK-8 assay at 24 h, 48 h, and 72 h, respectively. Although there was no difference in cell viability at 24 h in response to each treatment, at 48 h, high-glucose (HG) treatment markedly decreased MP5 cell viability relative to treatment with either normal glucose levels (NG) or mannitol (MA). Viability could be restored by cotreatment with HG and ADSC-derived EVs. No significant differences in MP5 cell viability were seen between the 48- and 72-h time points in response to treatment (Fig. 4B); therefore, the 48-h time point was chosen for all subsequent experiments. Flow cytometry was used to detect apoptosis at 48 h after the same treatments described above. The results showed that compared with treatment with either NG or MA, HG induction markedly increased MP5 cell apoptosis, whereas cotreatment of HG and ADSC-derived EVs exhibited reduced cell apoptosis versus HG treatment alone (Fig. 4C). In addition, Western blot analysis was used to detect the expression of apoptosis-related proteins in MP5 cells after 48 h of each treatment. Treatment with HG significantly decreased the protein expression of Bcl-2, whereas increasing that of caspase-3, cleaved caspase-3, and Bax compared with either NG or MA and cotreatment of HG and ADSC-derived EVs increased Bcl-2 protein expression and de-creased that of caspase-3, cleaved caspase-3, and Bax (Fig. 4D). These results suggested that EVs from ADSCs are able to suppress HG-mediated injury of podocytes in vitro. The expression of miR-26a-5p in HG-induced MP5 cells was measured by RT-qPCR after treatment with ADSC-derived EVs or PBS. miR-26a-5p expression was significantly lower after induction with HG than with treatment with NG or MA, but it was notably increased by treatment with ADSC-derived EVs containing miR-26a-5p (Fig. 5A). To confirm that ADSC-derived EVs can transfer miR-26a-5p into MP5 cells, Cy3-miR-26a-5p mimic was transfected into ADSCs and then added to MP5 cell media. Fluorescence microscopy revealed that MP5 cells became noticeably red after treatment (Fig. 5B). These results demonstrate that ADSC-derived EVs transfer miR-26a-5p into MP5 cells. To determine the effect of miR-26a-5p transferred by ADSC-derived EVs on HG-treated MP5 cells, HG-treated MP5 cells were transfected with either miR-26a-5p mimic, ADSC-derived EVs, or ADSC-derived EVs treated with miR-26a-5p inhibitor. Addition of either miR-26a-5p mimic or ADSC-derived EVs increased the expression of miR-26a-5p in HG-treated MP5 cells, whereas treatment of MP5 cells with ADSC-derived EVs containing miR-26a-5p inhibitor caused a decline in miR-26a-5p expression (Fig. 5C). Next, MP5 cell viability was assessed using the CCL-8 assay, and apoptosis was measured by flow cytometry. Under HG conditions, treatment with either miR-26a-5p mimic or ADSC-derived EVs markedly improved cell viability and suppressed apoptosis of MP5 cells, whereas ADSC-derived EVs treated with miR-26a-5p inhibitor led to decreased cell viability and increased apoptosis (Fig. 5, D and E). In addition, Western blot analysis was used to detect apoptosis-related proteins in MP5 cells after each treatment. Treatment with miR-26a-5p mimic or ADSC-derived EVs resulted in increased Bcl-2 protein expression accompanied by a notable decrease in Bax, cleaved caspase-3, and caspase-3 expression. In contrast, ADSC-derived EVs treated with miR-26a-5p inhibitor exhibited markedly decreased Bcl-2 protein expression, whereas the expression of Bax, cleaved caspase-3, and caspase-3 was significantly elevated (Fig. 5F). These results indicate that delivery of miR-26a-5p into MP5 cells by ADSC-derived EVs inhibits HG-induced apoptosis. The website TargetScan (RRID:SCR_010845) was able to predict specific binding sites between TLR4 and miR-26a-5p at nucleotides 4477–4484 (Fig. 6A). A Dual-Luciferase reporter gene assay was used to confirm that TLR4 was a target of miR-26a-5p. Cotransfection of miR-26a-5p mimic and TLR4-3'UTR-WT resulted in a marked decrease in fluorescence intensity compared with the mimic negative control (NC) + TLR4-3'UTR-WT cotransfection; however, fluorescence intensity was unchanged in the presence of mimic NC + TLR4-3'UTR-MUT or miR-26a-5p mimic + TLR4-3'UTR-MUT (Fig. 6B). RT-qPCR of MP5 cells transfected with miR-26a-5p mimic or inhibitor resulted in a noticeably increased miR-26a-5p expression and a significantly decreased TLR4 expression, whereas opposite effects were detected after treatment with miR-26a-5p inhibitor (Fig. 6, C and D). Western blot analysis revealed that HG treatment of MP5 cells significantly increased TLR4 protein expression compared with treatment with NG or MA treatment, and this could be reversed by treatment with EVs from ADSCs (Fig. 6E). This suggests that miR-26a-5p transferred by ADSC-derived EVs could directly target the TLR4 gene. To detect the effect of the TLR4 gene regulated by ADSC-derived EVs on HG-induced MP5 cells, HG-treated MP5 cells were overexpressed with TLR4 and then treated with ADSC-derived EVs. First, Western blot analysis was used to detect the cellular protein expression of TLR4 upon each treatment. It was found that under HG conditions, compared with pcDNA-3.1 treatment, pcDNA-TLR4 significantly upregulated the protein expression of TLR4, which was reversed by treatment of pcDNA-TLR4- and ADSC-derived EVs (Fig. 6F). Subsequently, CCK-8 assay was performed to detect MP5 cell viability, and flow cytometry was used to detect cell apoptosis. The results displayed that under HG conditions, relative to pcDNA-3.1 treatment, pcDNA-TLR4 significantly inhibited the protein cell viability while promoting cell apoptosis; however, promoted cell viability and inhibited cell apoptosis were detected after treatment with pcDNA-TLR4- and ADSC-derived EVs (Fig. 6G and H). In addition, Western blot analysis was used to detect apoptosis-related proteins in HG-treated MP5 cells after treatment with each plasmid. pcDNA-TLR4 treatment markedly decreased Bcl-2 protein expression while significantly increasing cleaved caspase-3, caspase-3, and Bax protein expression relative to that of pcDNA-3.1 treatment. The opposite results were found after the treatment of HG-induced MP5 cells transfected with pcDNA-TLR4 and exposed to EVs from ADSCs containing miR-26a-5p (Fig. 6I). Combined with the above results, we concluded that miR-26a-5p delivered by ADSC-derived EVs targets TLR4, inhibiting HG-induced MP5 cell apoptosis. Subsequently, we explored the role of ADSC-derived EVs in the progression of DN via the regulation of the NF-κB pathway and VEGFA. We first investigated whether miR-26a-5p carrying EVs from ADSCs could inhibit the NF-κB pathway by downregulating TLR4 expression. After TLR4 overexpression, MP5 cells induced by HG were further treated with ADSC-derived EVs. Immunofluorescence staining revealed that HG treatment of MP5 cells resulted in p65 localization to the nucleus. EVs from ADSCs were able to reduce p65 nuclear localization, keeping more p65 in the cytoplasm. The effects of EVs could be negated by the overexpression of TLR4, which led to the nuclear localization of p65 protein again (Fig. 7A). Western blot analysis compared the protein expression of NF-κB pathway-related proteins IKKβ, IκBα, and p65 and the extent of their phosphorylation in the cells after each treatment. Treatment with EVs from ADSCs and pcDNA-3.1 control plasmid led to significantly decreased protein expression of IKKβ, IκBα, and p65 and the extent of their phosphorylation during HG induction, whereas each of these proteins was upregulated by ADSC-derived EVs cotreated with pcDNA-3.1-TLR4 (Fig. 7B). To verify whether EVs from ADSCs can inhibit apoptosis associated with HG treatment by inhibiting the NF-κB pathway, HG-induced MP5 cells were treated with ADSC-derived EVs and the NF-κB pathway activator, phorbol-12-myristate-13-acetate (PMA; 1 μg/ml). Western blot analysis indicated that PMA treatment significantly increased the protein expression of IKKβ, IκBα, p65, and VEGFA, as well as the extent of their phosphorylation in HG-induced MP5 cells compared with that of treatment with DMSO. Combined treatment with PMA and ADSC-derived EVs significantly decreased all these indicators compared with PMA treatment alone (Fig. 7C). PMA treatment also notably decreased cell viability, as measured by CCK-8 assay, and increased cell apoptosis, as measured by flow cytometry, relative to treatment with DMSO. This effect was reversed by treatment with PMA and ADSC-derived EVs combined (Fig. 7, D and E). HG-induced MP5 cells were further treated with ADSC-derived EVs after overexpression of VEGFA. VEGFA protein expression was significantly decreased after treatment with ADSC-derived EVs, whereas further overexpression of VEGFA upregulated the VEGFA protein expression (Fig. 7F). HG-induced MP5 cells treated with ADSC-derived EVs plus pcDNA-3.1 displayed increased viability and decreased apoptosis of MP5 cells compared with that of cells treated with PBS. This was reversed by treatment with ADSC-derived EVs and pcDNA-3-VEGFA (Fig. 7G). These data suggest that ADSC-derived EVs inhibit TLR4 expression, resulting in the downregulation of NF-κB/VEGFA and inhibition of HG-induced MP5 cell injury. To explore the effects of miR-26a-5p delivered by ADSC-derived EVs in a mouse model of DN, ADSC-derived EVs and/or miR-26a-5p antagomir were injected into C57BL/KsJ db/db mice via the tail vein. Mice were then observed for changes in urine protein, Scr, and BUN levels, and the histopathological changes of mice were detected using PAS staining. As illustrated, diabetic mice treated with PBS and antagomir NC displayed accumulated extracellular matrix in their kidney tissues, viability of mesangial cells, thickening of basement membrane, and nuclear lamina dispersion, with increased proteinuria in the lumen, hyaline degeneration, and severe vacuolar degeneration of renal tubular epithelial cells. ADSC-derived EVs and antagomir NC treatment led to decreased levels of urine protein, Scr, and BUN in DN modeled mice and alleviated histopathological changes compared with those of PBS and antagomir NC treatment. Treatment with ADSC-derived EVs and miR-26a-5p antagomir treatment markedly increased the levels of urine protein, Scr, and BUN while aggravating the histopathological changes in the mice compared with treatment with ADSC-derived EVs and antagomir NC (Fig. 8, A and B). TUNEL staining showed that treatment with both ADSC-derived EVs and antagomir NC significantly reduced cell apoptosis compared with that of DN mice treated with PBS and antagomir NC. Apoptosis was notably increased in DN mice treated with both ADSC-derived EVs and miR-26a-5p antagomir compared with treatment with ADSC-derived EVs and antagomir NC (Fig. 8C). RT-qPCR was used to analyze miR-26a-5p and TLR4 expression in the renal tissues of mice in response to different treatments. Treatment with ADSC-derived EVs and antagomir NC significantly increased miR-26a-5p expression and decreased TLR4 expression compared with PBS plus antagomir NC treatment; however, treatment with ADSC-derived EVs plus miR-26a-5p antagomir decreased miR-26a-5p expression and increased TLR4 expression compared with treatment with ADSC-derived EVs and antagomir NC (Fig. 8D). Western blot analysis was used
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