Improvement of radiotherapy-induced lacrimal gland injury by induced pluripotent stem cell-derived conditioned medium via MDK and inhibition of the p38/JNK pathway

Abstract

Radiation therapy is the most widely used and effective treatment for orbital tumors, but it causes dry eye due to lacrimal gland damage. Induced pluripotent stem cell-derived conditioned medium (iPSC-CM) has been shown to rescue different types of tissue damage. The present study investigated the mechanism of the potential radioprotective effect of IPS cell-derived conditioned medium (iPSC-CM) on gamma-irradiation-induced lacrimal gland injury (RILI) in experimental mice. In this study, we found that iPSC-CM ameliorated RILI. iPSC-CM markedly decreased radiotherapy induced inflammatory processes, predominantly through suppressing p38/JNK signaling. Further signaling pathway analyses indicated that iPSC-CM could suppress Akt (Protein Kinase B, PKB) phosphorylation. High levels of midkine (MDK) were also found in iPSC-CM and could be involved in lacrimal gland regeneration by promoting cell migration and proliferation. Thus, our study indicates that inhibiting the p38/JNK pathway or increasing the MDK level might be a therapeutic target for radiation-induced lacrimal gland injury.

Figure 1

Radiotherapy-impaired lacrimal secretion and induced lacrimal gland injury in vivo. (A) Gross observation indicating normal and radiotherapy-treated lacrimal glands and the restorative effect of iPSC-CM or MFCM (mouse fibroblasts-derived conditioned medium) on irradiated lacrimal glands; (B) HE staining of irradiated lacrimal glands. Scale bar = 25 µm. One arrow means inflammatory cells, two arrows means the apoptotic acinar cells and three arrows means normal acinar cells; (C–E) Scintigraphic assessment of lacrimal gland secretion function of normal, normal + CM, RT, RT + CM, or RT + MFCM (CM: iPSC-CM, RT: radiotherapy, MFCM: mouse fibroblasts conditioned medium); N = 5. The values are the means ± SD. * p < 0.01.

Figure 2

Supplementary figure. (A) The mice underwent sequential scintigraphy in a prone position with frontal projection of the head using a four-head camera; (B) Scintigraphic data analyzed by software; (C–G) Time–activity curve of 99 mTc pertechnetate in the major lacrimal glands of mice in the five groups.

Figure 3

iPSC-CM suppressed the RILI-associated inflammatory response. (A) Immunohistochemical staining for PAI1 in iPSC-CM treated RILI and normal mouse lacrimal glands. Scale bar = 25 µm; (B) Immunohistochemical staining for HMGB1 in iPSC-CM treated RILI and normal mouse lacrimal glands. Scale bar = 25 µm; (C,D) Quantification of the mean density of immunohistochemical staining of these sections (CM: iPSC-CM, RT: radiotherapy, MFCM: mouse fibroblasts conditioned medium). N = 5. Values are means ± SEM. * p < 0.01; (E) Neutrophils migrated into the injured gland sites revealed by the neutrophil counts and myeloperoxidase (MPO) assay. * p < 0.01.

Figure 4

Ultramicrostructural restoration by iPSC-CM. (A) A TEM image revealing the lacrimal gland ultramicrostructure in normal, normal + CM, RT, RT + CM, or RT + MFCM (normal + CM: non irradiated after iPSC-CM treatment, RT: radiotherapy, RT + CM: radiotherapy after iPSC-CM treatment, RT + MFCM: radiotherapy after mouse fibroblasts conditioned medium treatment); N = 5; (B) Western blot of the protein level of MDK, SFRP2, CXCL2 and LRRC15 in iPSC-CM and MFCM (MFCM: mouse fibroblasts conditioned medium); (C) Quantification of the relative density of western blot results. N = 5. Values are means ± SEM. ** p < 0.01, *** p < 0.001.

Figure 5

Recombinant MDK (Midkine) promotes LGE (Lacrimal gland epithelial) cell migration and proliferation. (A) Images of LGE cell scratch-wound healing assays. Scale bar = 250 µm; (B) Quantification of the reduced area; N = 5. The values are the means ± SEM. * p < 0.05, ** p < 0.01; (C) In vitro proliferation assay for LGE cells treated with MDK; N = 5. The values are the means ± SEM. * p < 0.05.

Figure 6

iPSC-CM suppresses the p38/JNK pathway. (A) Western blot (upper) and quantification (lower) of p38, JNK (B), Akt (C) and Erk (D) phosphorylation from the irradiation injury lacrimal gland showing the effects of administering MFCM, p38 inhibitor (SB203580, a p38 MAPK inhibitor with IC50 of 0.3–0.5 μM.) or iPSC-CM on p38 phosphorylation in irradiation injury lacrimal glands. Lacrimal injury was induced in mice receiving radiotherapy. Data shown are the mean ± SD of five independent experiments. In (A), (B) and (C) * p<0.05 vs. normal, normal + CM, RT + CM, or RT + SB203580; In (D) * p < 0.05 vs. normal + CM, RT, RT + CM, RT + MFCM, or RT + SB203580 (normal + CM: non-irradiated after iPSC-CM treatment, RT: radiotherapy, RT + CM: radiotherapy after iPSC-CM treatment, RT + MFCM: radiotherapy after mouse fibroblasts conditioned medium treatment); N = 5.