Int J Stem Cells.  2023 Feb;16(1):52-65. 10.15283/ijsc22014.

Mesenchymal Stem Cells Ameliorate Fibrosis by Enhancing Autophagy via Inhibiting Galectin-3/Akt/mTOR Pathway and by Alleviating the EMT via Inhibiting Galectin-3/Akt/GSK3β/Snail Pathway in NRK-52E Fibrosis

• Zhao Y
• Guo C
• Zeng L
• Li J
• Liu X
• Wang Y
• Zhao K
• Chen B

• KMID: 2539621
• DOI: http://doi.org/10.15283/ijsc22014

Abstract

Background and Objectives
Epithelial-Mesenchymal transition (EMT) is one of the origins of myofibroblasts in renal interstitial fibrosis. Mesenchymal stem cells (MSCs) alleviating EMT has been proved, but the concrete mechanism is unclear. To explore the mechanism, serum-free MSCs conditioned medium (SF-MSCs-CM) was used to treat rat renal tubular epithelial cells (NRK-52E) fibrosis induced by transforming growth factor-β1 (TGF-β1) which ameliorated EMT.
Methods and Results
Galectin-3 knockdown (Gal-3 KD) and overexpression (Gal-3 OE) lentiviral vectors were established and transfected into NRK-52E. NRK-52E fibrosis model was induced by TGF-β1 and treated with the SF-MSCs-CM for 24 h after modelling. Fibrosis and autophagy related indexes were detected by western blot and immunocytochemistry. In model group, the expressions of α-smooth muscle actin (α-SMA), fibronectin (FN), Galectin-3, Snail, Kim-1, and the ratios of P-Akt/Akt, P-GSK3β/GSK3β, P-PI3K/PI3K, P-mTOR/mTOR, TIMP1/MMP9, and LC3B-II/I were obviously increased, and E-Cadherin (E-cad) and P62 decreased significantly compared with control group. SF-MSCs-CM showed an opposite trend after treatment compared with model group. Whether in Gal-3 KD or Gal-3 OE NRK-52E cells, SF-MSCs-CM also showed similar trends. However, the effects of anti-fibrosis and enhanced autophagy in Gal-3 KD cells were more obvious than those in Gal-3 OE cells.
Conclusions
SF-MSCs-CM probably alleviated the EMT via inhibiting Galectin-3/Akt/GSK3β/Snail pathway. Meanwhile, Gal-3 KD possibly enhanced autophagy via inhibiting Galectin-3/Akt/mTOR pathway, which synergistically ameliorated renal fibrosis. Targeting galectin-3 may be a potential target for the treatment of renal fibrosis.

Keyword

MSCs; Fibrosis; NRK-52E; EMT; Galectin-3; Autophagy

Figure

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  Fig. 1 Identification of rat bone marrow-derived MSCs. The appearance of bone marrow-derived MSCs is a typical spindle shape. Immunophenotypic characterization of MSCs was detected by flow cytometry. MSCs related markers were positive for CD29 and CD90 and were negative for CD106 and CD45.

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  Fig. 2 Effects of SF-MSCs-CM on TGF-β-induced NRK-52E fibrosis at different time points. (A) The expression of α-SMA and Gal-3 in NRK-52E cells induced by TGF-β1 was detected by Western Blot after SF-MSCs-CM treatment. (B) Quantification of α-SMA. (C) Quantification of Gal-3. Results were normalized relative to the expression of GAPDH. N=3 (per group). Data are presented as mean±SD and analyzed by two-way ANOVA followed by Tukey post hoc testing. *p<0.05, vs. control group, #p<0.05, vs. TGF-β1＋SF-MSCs-CM (24 h) group, ●p<0.05, vs. TGF-β1＋SF-MSCs-CM (48 h) group, ▲p<0.05, vs. TGF-β1 ＋SF-DMEM (24 h) group, ◆p<0.05, vs. TGF-β1＋SF-DMEM (48 h) group.

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  Fig. 3 Effects of SF-MSCs-CM on TGF-β1-induced NRK-52E cell fibrosis. Compared with TGF-β1 group, these fibrosis related indicators were notably downregulated in TGF-β1＋SF-MSCs-CM group. Results were normalized relative to the expression of GAPDH. N=3 (per group). Data are presented as mean±SD and analyzed by two-way ANOVA followed by Tukey post hoc testing. *p<0.05, vs. control group, ★p<0.05, vs. TGF-β1＋SF-MSCs-CM group, ▲p<0.05, vs. TGF-β1＋SF-DMEM group, ●p<0.05, vs. SF-MSCs-CM group.

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  Fig. 4 Effects of SF-MSCs-CM on TGF-β1-induced NRK-52E cell autophagy at different time points. SF-MSCs-CM treatment upregulated the ratio of LC3B-II/I compared with the TGF-β1 group, accompanied by down-regulation of P62, SF-DMEM treatment after TGF-β1 treatment significantly downregulate the ratio of LC3B-II/I meanwhile upregulated the expression of P62 compared with the TGF-β1＋SF-MSCs-CM group. However, the ratio of LC3B-II/I of SF-MSCs-CM treatment for 48 h after TGF-β1 treatment could have a downward trend compared with 24 h, P62 does the opposite. (A) Quantification of LC3B-II/I. (B) Quantification of P62. Results were normalized relative to the expression of GAPDH. N=3 (per group). Data are presented as mean±SD and analyzed by two-way ANOVA followed by Tukey post hoc testing. *p<0.05, vs. normal group, #p<0.05, vs. TGF-β1＋SF-MSCs-CM group (24 h), ●p<0.05, vs. SF-MSCs-CM group (48 h), ▲p<0.05, vs. TGF-β1＋SF-DMEM group (24 h), ◆p<0.05, vs. TGF-β1＋SF-DMEM group (48 h), &p<0.05, vs. SF-MSCs-CM group (24 h).

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  Fig. 5 The expression of Gal-3 in NRK-52E cells was detected by fluorescence microscope and western blot. Results were normalized relative to the expression of GAPDH. Results were normalized relative to the expression of GAPDH. N=3 (per group). Data are presented as mean±SD and analyzed by two-way ANOVA followed by Tukey post hoc testing. *p<0.05, **p<0.001 vs. control group. Scale bar (A)=200 μm, (B)=400 μm.

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  Fig. 6 Effects of SF-MSCs-CM on TGF-β 1-induced Gal-3 KD NRK-52E cells and Gal-3 OE NRK-52E cells fibrosis. The black and white bands illustrating the indicators identified through Western Blot (A), and statistical analysis of the ratio relative to GAPDH, including α-MSA (B), the ratio of TIMP1/MMP9 (C), FN (D), E-Cadherin (E), KIM-1 (F), Galectin-3 (G), the ratio of P-Akt/Akt (H) and P-GSK3β/GSK3β1 (I), and Snail (J). SF-MSCs-CM treatment significantly downregulated these indexes more surely than SF-DMEM treatment after TGF-β 1 treatment, except the expression of E-cad, which was upregulated. In the Gal-3 OE group, the indexes presented similar trends to those of the Gal-3 KD group, but higher than those of the same subgroups in the Gal-3 KD group. Results were normalized relative to the expression of GAPDH. N=3 (per group). Data are presented as mean±SD and analyzed by two-way ANOVA followed by Tukey post hoc testing. *p<0.05, vs. control group, ★p<0.05, vs. TGF-β 1＋SF-MSCs-CM group, ▲p<0.05, vs. TGF-β 1＋SF-DMEM group, ●p<0.05, vs. SF-MSCs-CM group; compared Gal-3 KD NRK-52E cells with Gal-3 OE NRK-52E cells, #p<0.05, vs. normal group, @p<0.05, vs. TGF-β 1 group, &p<0.05, vs. TGF-β 1＋SF-MSCs-CM group, ◆p<0.05, vs. TGF-β 1＋SF-DMEM group, ^p<0.05, vs. SF-MSCs-CM group.

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  Fig. 7 Immunocytochemical staining of E-Cadherin and α-SMA in Gal-3 KD and Gal-3 OE NRK-52E cells. Gal-3 KD reduced the expre-ssion of α-SMA and increased the expression of E-Cadherin in NRK-52E cells, but an opposite trend appea-red in Gal-3 OE. TGF-β1 indeed increased the expression of α-SMA and decreased the expression of E-Cadherin in Gal-3 OE cell, which was more than in Gal-3 KD cells. The treatment of SF-MSCs-CM after TGF-β1 treatment reduced the expression of α-SMA and raised the expression of E-Cadherin in both Gal-3 KD cells and Gal-3 OE cells, but more obvious in Gal-3 KD cells than in Gal-3 OE cells. SF-DMEM treatment also downregulated the expression of α-SMA, worse than SF-MSCs-CM group. Bar=100 μm.

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  Fig. 8 Expression of autophagy related proteins. (A∼F), Whether in Gal-3 KD cells or in Gal-3 OE cells, SF-MSCs-CM treatment enhanced autophagy and simultaneously reduced fibrosis after TGF-β1 treatment. SF-DMEM treatment also appear the similar phenomenon, but less than those in TGF-β1＋SF-MSCs-CM group, especially in Gal-3 OE cells. Results were normalized relative to the expression of GAPDH. N=3 (per group). Data are presented as mean±SD and analyzed by two-way ANOVA followed by Tukey post hoc testing. *p<0.05, vs. control group, ★p<0.05, vs. TGF-β1＋SF-MSCs-CM group, ▲p<0.05, vs. TGF-β1 ＋SF-DMEM group, ●p<0.05, vs. SF-MSCs-CM group; compared Gal-3 KD NRK-52E cells with Gal-3 OE NRK-52E cells, #p<0.05, vs. normal group, @p<0.05, vs. TGF-β1 group, &p<0.05, vs. TGF-β1＋SF-MSCs -CM group, ◆p<0.05, vs. TGF-β1＋SF-DMEM group, ^p<0.05, vs. SF-MSCs-CM group.

Reference

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