Synergism of calycosin and bone marrow-derived mesenchymal stem cells to combat podocyte apoptosis to alleviate adriamycin-induced focal segmental glomerulosclerosis

Figure 1 Mesenchymal stem cells pretreated with calycosin enhance the protective effect of mesenchymal stem cells on podocyte injury in adriamycin-induced focal segmental glomerulosclerosis mice. A: Mice received adriamycin injections through the tail vein at week 6, were injected with Dulbecco’s modified eagle medium, mesenchymal stem cells (MSCs), calycosin (CA), and MSCs pretreated with CA (MSCs^CA) at week 10, respectively, and were sacrificed at week 14; B: Levels of albumin/creatinine ratio in urine (n = 6), ^aP < 0.05, ^bP < 0.001; C: Pathological changes in the kidneys of mice examined by hematoxylin-eosin staining. Typical glomeruli are indicated by black boxes and enlarged to the next row. Bar = 50 μm; D: Changes in the kidneys of mice examined by podocin immunofluorescence staining. Glomeruli are indicated by white boxes and enlarged to the next row. Bar = 50 μm; E: Relative (podocin/GAPDH) mRNA expression analyzed by real-time quantitative polymerase chain reaction. Data are expressed as the mean ± SD (n = 6). ^aP < 0.05, ^bP < 0.001; F and G: Relative protein levels (podocin/GAPDH) detected by Western blot. Data are expressed as the mean ± SD (n = 3). ^aP < 0.05, ^bP < 0.001. NC: Normal control; ADR: Adriamycin; DMEM: Dulbecco’s modified eagle medium; MSCs: Mesenchymal stem cells; CA: Calycosin; MSCs^CA: Mesenchymal stem cells pretreated with calycosin.

Figure 2 Calycosin pretreatment enhances the ability of mesenchymal stem cells to inhibit apoptosis in adriamycin-induced focal segmental glomerulosclerosis mice. A-C: Protein expression levels of Bax and Bcl-2 in the kidneys measured by Western blot and normalized to control. Data are expressed as the mean ± SD (n = 3). ^aP < 0.05, ^bP < 0.001; D: Apoptosis in each group as determined by TUNEL assay. Bar = 50 μm. NC: Normal control; ADR: Adriamycin; MSCs: Mesenchymal stem cells; CA: Calycosin; MSCs^CA: Mesenchymal stem cells pretreated with calycosin.

Figure 3 P-Smad3 is upregulated in podocytes of adriamycin-induced focal segmental glomerulosclerosis mice and reversed after treatment with mesenchymal stem cells pretreated with calycosin. A and B: Protein expression levels of p-Smad3 and Smad3 detected using Western blot and normalized to control. Data are expressed as the mean ± SD (n = 3). ^bP < 0.001; C: Immunohistochemistry staining for p-Smad3 in mouse glomeruli, which are indicated by red boxes and enlarged to the next row. Bar = 50 μm. NC: Normal control; ADR: Adriamycin; MSCs: Mesenchymal stem cells; CA: Calycosin; MSCs^CA: Mesenchymal stem cells pretreated with calycosin.

Figure 4 Calycosin pretreatment enhances the ability of mesenchymal stem cells to ameliorate the injury of adriamycin-stimulated mouse podocyte cells in vitro. A: Podocin expression in each group as determined by immunofluorescence staining. Bar = 50 μm; B: Analysis of relative (podocin/GAPDH) mRNA expression by real-time quantitative polymerase chain reaction. Data are expressed as the mean ± SD (n = 3). ^bP < 0.001; C and D: Protein expression levels of podocin detected by Western blot and normalized to control. Data are expressed as the mean ± SD (n = 3). ^aP < 0.05, ^bP < 0.001. NC: Normal control; ADR: Adriamycin; MSCs: Mesenchymal stem cells; CA: Calycosin; MSCs^CA: Mesenchymal stem cells pretreated with calycosin.

Figure 5 Calycosin pretreatment enhances the ability of mesenchymal stem cells to inhibit apoptosis in adriamycin-stimulated mouse podocyte cells. A and B: Expression of Bax and Bcl-2 in each group as determined by immunofluorescence staining. Bar = 50 μm; C-E: Protein expression levels of Bax and Bcl-2 detected by Western blot and normalized to control. Data are expressed as the mean ± SD (n = 3). ^bP < 0.001. NC: Normal control; ADR: Adriamycin; MSCs: Mesenchymal stem cells; CA: Calycosin; MSCs^CA: Mesenchymal stem cells pretreated with calycosin.

Figure 6 Calycosin-pretreated mesenchymal stem cells improve adriamycin-induced podocyte apoptosis by targeting p-Smad3 expression. A and B: Protein expression levels of p-Smad3 and Smad3 in mouse podocyte cells (MPC5) detected by Western blot and normalized to control. Data are expressed as the mean ± SD (n = 3). ^aP < 0.05, ^bP < 0.001; C-G: Protein expression levels of Bax, Bcl-2, p-Smad3, and Smad3 in Smad3-overexpressing MPC5 cells detected by Western blot and normalized to control. Data are expressed as the mean ± SD (n = 3). ^bP < 0.001; H-L: Cell apoptosis detected by flow cytometry; M: Graphical abstract (created in BioRender.com). NC: Normal control; ADR: Adriamycin; MSCs: Mesenchymal stem cells; CA: Calycosin; MSCs^CA: Mesenchymal stem cells pretreated with calycosin; FSGS: Focal segmental glomerulosclerosis.