Combined Therapy With Adipose-Derived Mesenchymal Stem Cells and Ciprofloxacin Against Acute Urogenital Organ Damage in Rat Sepsis Syndrome Induced by Intrapelvic Injection of Cecal Bacteria

Abstract

We hypothesized that combined treatment with autologous adipose-derived mesenchymal stem cell (ADMSC) and ciprofloxacin is superior to ciprofloxacin only in reducing sepsis-induced urogenital organ damage and mortality in rat sepsis syndrome (SS) caused by intrapelvic injection of cecal bacteria (1.0 × 10(4) cells per milliliter; total, 5.0 ml). Male Sprague-Dawley rats (n = 60) equally divided into group 1 (sham-control), group 2 (SS), group 3 (SS-ADMSC [5.0 × 10(5) intravenously at 0.5, 6, and 18 hours after sepsis induction]), group 4 (SS-ciprofloxacin [3.0 mg/kg, b.i.d.] for 5 days), and group 5 (SS-ADMSC-ciprofloxacin) were sacrificed by day 5. Mortality rate and creatinine level were highest in group 2 and lowest in group 1 and significantly higher in groups 3 and 4 than those in group 5, but there was no difference between groups 3 and 4 (all p < .005). The kidney injury score, inflammatory biomarker expressions at protein (tumor necrosis factor-1α, nuclear factor-κB, matrix metallopeptidase-9, regulated on activation, normal T-cell expressed and secreted, interleukin-1β) and cellular (CD14+, migratory inhibitor factor positive, CD68+) levels in kidneys and urinary bladder were lowest in group 1 and highest in group 2, higher in group 4 than in groups 3 and 5, and higher in group 3 than in group 5 (all p < .001). Protein expressions of apoptosis (Bax, cleaved caspase 3 and poly[ADP-ribose] polymerase 1, p21 protein [Cdc42/Rac]-activated kinase 2) and oxidative stress (oxidized protein, NADPH oxidase (NOX)-1, NOX-2) in these organs showed an identical pattern compared with that of inflammation in all groups (all p < .001). In conclusion, ADMSC-assisted ciprofloxacin therapy offered an additional benefit by reducing acute urogenital organ damage in rat.

Significance: Autologous adipose-derived mesenchymal stem cell-assisted ciprofloxacin therapy offered an additional benefit by reducing acute urogenital organ damage in rats.

Keywords: Adipose-derived mesenchymal stem cells; Antibiotics; Inflammation; Sepsis syndrome; Urogenital organ damage.

Figure 1.

The flow cytometric analysis, mortality rate, and circulating level of creatinine by day 5 after SS induction (n = 16). (A–E): Flow cytometric analysis by day 14 after cell culturing. (F): Spindle-shape picture of the cells in culture plate, a typical appearance of mesenchymal stem cells (original magnification, ×100). (G): CD90+ and CD29+ cells were the two highest populations of ADMSCs. (H): Statistical analysis of mortality rate among five groups. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡). (I): Statistical analysis of circulating creatinine level; ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §). All statistical analyses were performed by one-way analysis of variance, followed by Bonferroni multiple-comparison post hoc test (n = 10 for group 1; n = 8 for group 2; n = 12 for groups 3 and 4; n = 14 for group 5). Symbols (∗, †, ‡, §) indicate significance (at the .05 level). Abbreviations: ADMSC, adipose-derived mesenchymal stem cell; Cipro, ciprofloxacin; SC, sham control; SS, sepsis syndrome.

Figure 2.

The protein expressions of inflammatory biomarkers in urinary bladder and kidney parenchyma by day 5 after sepsis induction (n = 8). Upper panel (protein expressions in bladder): (A): Protein expression of TNF-1α, ∗, p < .001 compared with other groups with different symbols (∗, †, ‡, §, ¶). (B): Protein expression of NF-κB. ∗, p < .001 vs. other groups with different symbols (∗, †, ‡, §, ¶) . (C): Protein expression MMP-9. ∗, p < .001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (D): Protein expression of RANTES. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (E): Protein expression of IL-1β. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). Lower panel (protein expression in kidney parenchyma): (F): Protein expression of TNF-1α. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (G): Protein expression of NF-κB. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (H): Protein expression of MMP-9. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (I): Protein expression of RANTES. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (J): Protein expression of IL-1β. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). All statistical analyses were performed by one-way analysis of variance, followed by Bonferroni multiple-comparison post hoc test. Symbols (∗, †, ‡, §, ¶) indicate significance (at the .05 level). Abbreviations: ADMSC, adipose-derived mesenchymal stem cell; Cipro, ciprofloxacin; IL, interleukin; MMP-9, matrix metalloproteinase-9; NF, nuclear factor; RANTES, regulated on activation, normal T-cell expressed and secreted; SC, sham control; SS, sepsis syndrome; TNF, tumor necrosis factor. The error bars are defined as standard error of the mean.

Figure 3.

Immunohistochemical (IHC) microscopic findings of inflammatory cells in urogenital organ by day 5 after sepsis induction (n = 8) (A–E): Microscopic finding (original magnification, ×200) of CD14+ cells in urinary bladder. (F): Score of CD14+ expression in bladder. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (G–K): IHC microscopic finding (original magnification, ×200) of CD14+ cells in kidney parenchyma. (L) Score of CD14+ in kidney. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶) . The scale bars in right lower corner represent 50 µm. All statistical analyses were performed by one-way analysis of variance, followed by Bonferroni multiple-comparison post hoc test. Symbols (∗, †, ‡, §, ¶) indicate significance (at the .05 level). Abbreviations: ADMSC, adipose-derived mesenchymal stem cell; Cipro, ciprofloxacin; SC, sham control; SS, sepsis syndrome.

Figure 4.

Immunofluorescent (IF) microscopic identification of inflammatory cells in urinary bladder by day 5 after sepsis induction. (A–E): IF microscopic (original magnification, ×200) finding of macrophage MIF+ cells in urinary bladder. The MIF+ cells (green) and implanted ADMSCs (red) in smaller dotted-line square were magnified to larger dotted-line square in (C) and (E). (F): Percentage of MIF+ cells in bladder. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (G–K): IF microscopic (original magnification, ×200) finding of CD68+ cells in urinary bladder. The CD68+ cells (green) and implanted ADMSCs (red) in smaller dotted-line square were magnified to larger dotted-line square in (I) and (K). (L): Percentage of CD68+ cells in bladder. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). The scale bars in right lower corner represent 50 µm. All statistical analyses were performed by one-way analysis of variance, followed by Bonferroni multiple-comparison post hoc test. Symbols (∗, †, ‡, §, ¶) indicate significance (at the .05 level). Abbreviations: ADMSC, adipose-derived mesenchymal stem cell; Cipro, ciprofloxacin; MIF, migratory inhibitor factor; SC, sham control; SS, sepsis syndrome.

Figure 5.

Immunofluorescent (IF) microscopic identification of inflammatory cells in kidney parenchyma by day 5 after sepsis induction. (A–E): IF microscopic (original magnification, ×200) finding of macrophage MIF+ cells in kidney parenchyma. The MIF+ cells (green) and implanted ADMSCs (red) in smaller dotted-line square were magnified to larger dotted- line square in (C) and (E). (F): Percentage of MIF+ cells in kidney. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (G–K): IF microscopic (original magnification, ×200) finding of CD68+ cells in kidney parenchyma. The CD68+ cells (green) and implanted ADMSCs (red) in smaller dotted-line square were magnified to larger dotted-line square in (I) and (K). (L): Percentage of CD68+ cells in kidney. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). The scale bars in right lower corner represent 50 µm. All statistical analyses were performed by one-way analysis of variance, followed by Bonferroni multiple-comparison post hoc test. Symbols (∗, †, ‡, §, ¶) indicate significance (at the .05 level). Abbreviations: ADMSC, adipose-derived mesenchymal stem cell; Cipro, ciprofloxacin; MIF, migratory inhibitor factor; SC, sham control; SS, sepsis syndrome.

Figure 6.

The protein expressions of apoptosis and reactive oxygen species in urinary bladder and kidney parenchyma by day 5 after sepsis induction. Upper panel (protein expressions in the bladder): (A): Protein expression of mitochondrial Bax. ∗, p < .001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (B–D): Protein expressions of c-Casp 1, 2, and 3. ∗, p < .001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (E): Protein expression of c-PARP. ∗, p < .001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (F): Protein expression of PAK2. ∗, p < .001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (G): Protein expression of NOX-1. ∗, p < .001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (H): Protein expression of NOX-2. ∗, p < .001 vs. other groups with different symbols (∗, †, ‡, §, ¶). Lower panel (protein expressions in kidney parenchyma): (I): Protein expression of mitochondrial Bax. ∗, p < .001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (J–L): Protein expressions of c-Casp 1, 2, and 3. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (M): Protein expression of c-PARP, ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (N): Protein expression of PAK2. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (O): Protein expression of NOX-1. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (P): Protein expression of NOX-2. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). All statistical analyses were performed by one-way analysis of variance, followed by Bonferroni multiple-comparison post hoc test. Symbols (∗, †, ‡, §, ¶) indicate significance (at the .05 level). ADMSC, adipose-derived mesenchymal stem cell; c-Casp, cleaved caspases; c-PAK2, cleaved p21 activated kinase 2; c-PARP, cleaved poly(ADP-ribose) polymerase; Cipro, ciprofloxacin; COX IV, cytochrome c oxidase subunit IV; NOX, NADPH oxidase; SC, sham control; SS, sepsis syndrome. The error bars are defined as standard error of the mean.

Figure 7.

The protein expression of oxidative stress in urogenital organs and pathological assessment of the kidney parenchyma by day 5 after sepsis induction. (A): Protein expression of oxidized protein (i.e., oxidative index) in urinary bladder. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (B): Protein expression of oxidized protein in kidney parenchyma. ∗, p < .0001 vs. other groups with different symbols (∗, †, ‡, §, ¶). (Note: left and right lanes shown in the upper panel represent protein MW marker and control oxidized molecular protein Std, respectively). (C–G): Hematoxylin and eosin stain (original magnification, ×200) demonstrating significantly higher degree of loss of brush border in renal tubules (yellow arrows), tubular necrosis (green asterisk), and dilatation of Bowman capsule (blue arrows) in SS group than in other groups. (H): Analytical result of kidney injury score. ∗, p < .001 vs. other groups with different symbols (∗, †, ‡, §, ¶). All statistical analyses were performed by one-way analysis of variance, followed by Bonferroni multiple-comparison post hoc test. Symbols (∗, †, ‡, §, ¶) indicate significance (at the .05 level). ADMSC, adipose-derived mesenchymal stem cell; Cipro, ciprofloxacin; DNP, 1-3 dinitrophenylhydrazone; MW, molecular weight; SC, sham control; SS, sepsis syndrome; Std, standard.