Figure 6
The beneficial effects of hMSCs were dependent on its secreted TSG-6 in SAP in a mechanism involving oxidative stress, NLRP3 inflammasome and NF- κB signaling.
(A) Representative immunofluorescence co-staining of TSG-6 (red) and CD-44 (green) in NaT treated-PACs. The nuclei were visualized using DAPI (blue). Figures are representative of three independent experiments. Magnification ×630. (B,C) Representative immunofluorescence images of CD44 (green) in control PACs, CD44-siRNA-PACs, and scr-siRNA-PACs. Nuclei are stained with DAPI (blue). Magnification ×400. Western blot assay was also performed. Figures are representative of three independent experiments. (D) Evaluation of MDA and (E) SOD in serum. The levels of MDA (μmol/L) and SOD (U/mL) were evaluated in each group, (n = 8 or 12). (F) Transmitted light and DCFH-DA whole cell fluorescence (green) images of murine cells showing increases in ROS induced by NaT. In addition, quantitative detection in each group were detected via flow cytometry analysis. Evaluation of mRNA transcript levels of (G) NLRP3, (H) ASC, and (I) Casp-1for pancreatic tissue in each group. (J) Western blot analysis of ASC, Casp-1 20 subunit, pro-Casp-1 in PACs. β-actin were used as the internal references. (K) Representative pancreatic sections of immunofluorescent staining are shown for cytoplasmic and nuclear distribution of NF-κB. Original magnification: ×630. (L,M) Western blot analysis of NF-κB p65 in the nucleus of pancreatic acinar cells. Histone H3 and β-actin were used as the internal references for nuclear and whole cell proteins, respectively. (N) The increases of IL-1β, IL-6, and MCP-1 in culture supernatant of acinar cells incubated with or without macrophages (Raw264.7) stimulated by NaT were significantly reduced with 100 ng rhTSG-6. (O) Confluent monolayers of HUVECs labeled with Hoechst were grown on six-well plates and stimulated with SAP serum or SAP serum +100 ng rhTSG-6 for 24 h. CFSE fluorescent intensity of adherent THP-1 cell was measured and the final results were expressed as the fold induction of CFSE fluorescence compared with controls. Each value represents the mean ± standard deviation. N.S., not significant, *P < 0.05, **P < 0.01.
