Influences Of GDNF On Healing Of Mouse Corneal Epithelium By Reduction On Transformation Of M1/M2 Macrophages

PurposeTo observe the effects of glial derived neurotrophic factor(GDNF) on corneal epithelial healing and inflammation of diabetic mice during repair of corneal epithelium damage, through in vitro experiment(macrophages induced by mouse bone marrow stem cells(BMSC)) and in vivo experiments(mice with type I diabetes induced by streptozocin(STZ)); and to discuss the action mechanism. MethedsMouse BMSCs were cultured in vitro. Experimental grouping(1): control group, normal group, C57 group and C57+GDNF group. ELISA and confocal microscope were used to detect the differences of macrophage phagocytosis between the normal group and C57 group. Experimental grouping(2): control group, normal group, normal group + IL4 group, and normal group +IL4+GDNF group. The proportions of M1 macrophages transforming into M2 macrophages were detected. And PCR and ELISA were used to detect the changes of expressions of factors related to M1 and M2 macrophages. Experimental grouping(3): control group, macrophages M0 group, macrophage M0+ LPS group, and macrophage M0 + LPS + GDNF group. The proportions of M1 macrophages transforming into M2 macrophages were detected. And PCR and flow cytometry were used to detect the changes of expressions of factors related to M1 and M2 macrophages. Experimental grouping(4): control group, control group + LPS, control group + LPS +GDNF(10ng/ml) group, control group + LPS +GDNF(50ng/ml) group, and control group + LPS +GDNF(100ng/ml) group. The correlations between the transformation proportions of macrophages and different concentrations of GDNF were detected. PCR and ELISA were used to detect the proportions of related expression products of M1 macrophages and M2 macrophages.Mice with type C57/BL6 I diabetes induced by streptozocin(STZ) and normal C57/BL6 mice were selected. Corneal epithelium damage models were established in mice, and subconjunctival injection was adopted for medication. Experimental grouping: normal group, diabetes group, diabetes + GDNF group. Differences of endogenous GDNF expressions in corneal epithelium were detected in normal mice and diabetic mice; the influences of GDNF on healing rate of corneal epithelium damage in diabetic mice were observed; the influences of GDNF on inflammation of corneal epithelium damage in diabetic mice were detected; and the influences of GDNF on related products of M1 and M2 macrophages of corneal epithelium damage in diabetic mice were detected by PCR. ResultsExperimental studies on macrophages induced by mice BMSCs showed that compared with STZ group, phagocytosis of macrophages was obviously increased in STZ+GDNF group(P<0.05); experiments on anti-inflammatory factors of M2 macrophage showed that compared with normal+ IL-4 group, the expressions of anti-inflammatory factors of M2 macrophage were significantly increased in normal +IL-4+GDNF group(P<0.05). Experiments on inhibition of exogenous GDNF on expressions of inflammatory factors related to M1 macrophages showed that compared with the normal group, and the normal + LPS group, the expressions of related inflammatory factors were significantly decreased in the normal + LPS + GDNF group; Experiments on concentration dependence of GDNF on expressions of inflammatory factors related to M1 macrophages showed that the direction of GDNF inhibiting expressions of inflammatory factors related to M1 macrophages was little correlated with concentration of GDNF.Comparison on normal and STZ mice showed that healing rate of corneal epithelium after removal was obviously slower in the STZ group than in the normal group(P<0.05), while obviously faster in the diabetes + GDNF group than in the diabetes group(P<0.05). And after removal of mouse corneal epithelium, the 24 h expression of COX-2(a proinflammatory factor), the 48 h number of infiltration neutrophils, expressions of i NOS and IL-12(markers of M1 macrophages) and 72 h number of M1 macrophages were significantly higher in STZ group than in the normal group(P<0.05), while significantly decreased in STZ+GDNF group compared with STZ group(P<0.05). 72 hours after removal of corneal epithelium, the expressions of Arginase-1 and IL-10(markers of M2 macrophages) were obviously reduced in STZ group than in the normal group, while obviously increased in STZ + GDNF group compared with the STZ group(P<0.05). ConclusionGDNF promotes healing of damaged corneal epithelium in STZ mice, which may be related to primary generation of macrophages and transformation of M1 macrophages to M2 macrophages. GDNF also inhibits the expressions of inflammatory factors related to M1 macrophages and induces the transformation of M1 macrophage to M2 macrophages. In addition, GDNF can increase the anti-inflammatory factor expression of M2 macrophage and contribute to the enhancement of macrophage phagocytosis.