Effects of hydrogen sulfide on autophagy and angiogenesis of skin wound in diabetic rats.

To explore the effect and mechanism of hydrogen sulfide (H2S) on autophagy and angiogenesis in skin wound of diabetic rats. METHODS: Among 36 healthy 8-week-old male Sprague-Dawley rats, 12 rats were selected as control group, and the remaining rats were intraperitoneally injected with streptozotocin (STZ) to induce diabetic model and were randomly divided into diabetes mellitus (DM) group and NaHS (H2S donor) intervention (DM+NaHS) group, with 12 rats in each group. A skin trauma model was established by excising the skin of the back of rats in each group. The rats in DM+NaHS group were intraperitoneally injected with NaHS (56 µmol/kg), and the rats in control and DM groups were daily received the same volume of normal saline for 21 consecutive days. The healing of skin wound was measured on days 0, 7, 14 and 21 after operation. On the 21st day after surgery, the content of H2S in skin tissues was detected by C-7Az fluorescent probe, and the morphological changes and angiogenesis of wound tissues were observed by HE staining. The expression of CD31 was detected by immunofluorescence staining, and endothelial autophagy was detected by double staining of CD31 and beclin-1. The protein levels of cystathionine γ-lyase (CSE), CD31, microtubule-associated protein 1 light chain 3 (LC3), beclin-1, P62, Bcl-2, Bax, phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR) in wound tissues were determined by Western blot. Caspase-3 and propidium iodide (PI) staining was used to detect cell apoptosis, and apoptosis of vascular endothelial cells was determined with CD31 and TUNEL double immunofluorescence staining. RESULTS: Compared with DM group, the wound healing rate, H2S content and CSE protein expression were significantly increased in DM+NaHS group (P<0. 01), but still lower than those in control group (P<0.01). HE staining showed that the wound surface in DM group was thin and wide, with few capillary, while that in DM+NaHS group was thicker with lots of capillary and wound width was reduced. Compared with DM group, CD31 expression was markedly increased (P<0. 01), the fluorescence intensity of caspase-3 and PI was significantly decreased (P<0. 01, and CD31+/beclin-1+ as well as CD31+/TUNEL+ cells were decreased (P<0. 01) in DM+NaHS group. Western blot analysis showed that compared with DM group, the levels of beclin-1, Bax and LC3-II/LC3-I were significantly decreased (P<0. 01), while the levels of P62 and Bcl-2, as well as ratios of p-PI3K/PI3K, p-Akt/ Akt and p-mTOR/mTOR were significantly increased (P<0. 01) in DM+NaHS group. CONCLUSION: H2S can promote skin wound healing, which may be related to activation of PI3K/Akt/mTOR signaling pathway, inhibition of endothelial autophagy and apoptosis, and promotion of angiogenesis in diabetic rats. [ABSTRACT FROM AUTHOR]