miR-20a Overexpression in Adipose-Derived Mesenchymal Stem Cells Promotes Therapeutic Efficacy in Murine Lupus Nephritis by Regulating Autophagy

Abstract

Objective. Lupus nephritis is the most common and severe complication of systemic lupus erythematosus. The aim of our study was to investigate the efficacy of miR-20a overexpressing adipose-derived stem cell (ADSC) transplantation in murine lupus nephritis (LN) and explore potential molecular mechanisms. Methods. Mouse ADSCs were transfected with a miR-20a lentiviral vector to obtain miR-20a overexpression ADSCs (miR-20a-ADSCs). We first observed the influence of miR-20a on ADSC viability and apoptosis in vitro. B6.MRL/lpr mice were administered ADSC/miR-20a-ADSC intravenously every week from age 30 to 33 weeks, and the lupus and normal control groups received PBS on the same schedule. Results. miR-20a expression increased in miR-20a-ADSC-derived exosomes, and miR-20a overexpression promoted ADSC proliferation and inhibited apoptosis. Compared with ADSCs, miR-20a-ADSC treatment significantly improved serologic and histologic abnormalities, as evidenced by reduced serum creatinine, anti-dsDNA antibody, 24 h urine protein levels, nephritis scores, and C3/IgG deposits. Furthermore, miR-20a-ADSC treatment resulted in downregulated Akt, mTOR, and p62 expression and upregulated miR-20a, Beclin 1, and LC3 II/I expression compared with ADSC treatment. After treatment with miR-20a-ADSC, a significant increase in the number of autophagosomes within podocytes was observed, along with upregulated expression of podocin and nephrin, compared with the ADSC group. Conclusions. miR-20a-ADSC transplantation prevents the development of lupus nephritis and significantly ameliorates already-established disease, and its mechanism is related to autophagy by targeting the miR-20a-regulated mTOR pathway.

Potential Therapeutic Application and Mechanism of Action of Stem Cell-Derived Extracellular Vesicles (EVs) in Systemic Lupus Erythematosus (SLE)

“…The EVs also had a positive effect on the downregulation of the secretion of proinflammatory cytokines TNF-α, IL-6, IL-16 IL-12, IFN-γ, IL-1β, IL-17, and GM-CSF, indicating depolarisation of macrophages into the M1 phenotype [1,[43][44][45][46][47][48][49][50][51]. However, in the study by Zhao et al [45], IL-6 (pro-inflammatory cytokine) levels showed signs of upregulation due to the possibility of paracrine effects of MSCs that elevate IL-6 levels.…”

“…Some studies have isolated EVs from respective MSCs using isolation kits. One study indicated that EVs from adipose-derived mesenchymal stem cells (ADSCs) were isolated using an isolation kit (ExoQuick-TC)-System Bioscience, Palo Alto, CA, USA, where the isolation reagent was added into the cell pellet overnight and centrifuged to obtain the EVs [48]. Another study indicated that the EVs were isolated from a Cell Culture Media Exosome Purification Mini Kit-Norgen Biotek, Thorold, ON, Canada [49].…”

“…The Western blot assay is used to determine specific protein markers related to EVs, which are commonly identified as CD9, CD36, and endoplasmic reticulum-oriented calnexin [43]. Other protein markers found in the selected studies that were used to identify and characterise EVs include CD63, Alix, CD81, CD63, GAPDH, and TSG101 [43][44][45][46][47][48][49][50][51]. Characterisation of EVs based on morphology was conducted using a transmission electron microscope and was recorded in a few of the studies.…”

“…In the study of Chen et al [43] and Chen et al [46], the mice were injected with 100 µL of 0.2 mg/mL EVs derived from hUC-MSCs via intravenous injection through the tail vein every 2 days for 14 days. Instead of 2 days, the study carried out by Wei et al [48] reported 2 × 10 5 cells per 10 g animal weight of ADSC/miR-20a per 150 µL PBS solution weekly for 14 days. Instead of multiple injections of EVs, a study by Sun et al [47] recorded single doses of EVs of 200 µg based on the protein concentration that were administered.…”

Mesenchymal Stem Cell-Derived Exosomes: Toward Cell-Free Therapeutic Strategies in Chronic Kidney Disease

“…However, despite the efficacy and clinical potential for therapeutic application in inflammatory glomerular disease indicated by these studies, there are few publications applying MSC-Exos directly in LN animal models or in human patients. Recently, Wei et al reported that miR-20a-containing exosomes are responsible for the alleviation of LN in the mouse lupus model through enhancing autophagy ( 137 ). In another study by Chen et al, UC-MSC-Exos attenuated SLE-associated diffuse alveolar hemorrhage (DAH) by regulating macrophage polarization in murine lupus ( 138 ).…”