硫化氢对糖尿病大鼠皮肤创面自噬和血管形成的影响

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

目的:探讨硫化氢(H2S)对糖尿病(DM)大鼠皮肤创面自噬及血管形成的影响及机制。方法:健康8周龄雄性SD大鼠36只,随机选取12只作为正常对照(control)组,余大鼠腹腔注射链脲佐菌素(STZ)诱导糖尿病模型,将建模成功24只大鼠随机分为糖尿病模型(DM)组和NaHS(H2S供体)干预(DM+NaHS)组,每组12只。手术切除各组大鼠背部皮肤建立皮肤创伤模型,DM+NaHS组大鼠每日腹腔注射NaHS(56μmol/kg),DM组和control组大鼠每日腹腔注射等量生理盐水,连续注射21 d。手术第0、7、14、21天检测皮肤创面愈合情况。手术第21天,取皮肤创面组织,应用H2S荧光探针C-7Az检测皮肤组织H2S含量;HE染色观察创面组织形态学变化及血管形成情况;免疫荧光染色检测创面组织CD31表达;采用CD31和beclin-1免疫荧光双重染色检测血管内皮细胞自噬情况;Western blot检测创面组织胱硫醚γ-裂解酶(CSE)、CD31、微管相关蛋白1轻链3(LC3)、beclin-1、P62、Bcl-2、Bax、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(PKB/Akt)和哺乳动物雷帕霉素靶蛋白(mTOR)蛋白表达;碘化丙啶(PI)及caspase-3免疫荧光染色检测创面组织细胞凋亡,通过CD31和TUNEL荧光双重染色检测创面组织血管内皮细胞凋亡情况。结果:与DM组相比,DM+NaHS组皮肤创面愈合率、H2S含量及CSE蛋白表达显著升高(P<0.01),但低于control组水平(P<0.01)。HE染色显示,DM组创面表层薄,毛细血管少,创面宽;DM+NaHS组创面表层增厚,可见大量毛细血管,创面宽度减小。与DM组相比,DM+NaHS组创面组织CD31表达显著增加(P<0.01),caspase-3和PI荧光强度显著降低(P<0.01),CD31+/beclin-1+和CD31+/TUNEL+阳性细胞显著减少(P<0.01)。Western blot结果显示,与DM组相比,DM+NaHS组LC3-II/LC3-I、beclin-1和Bax表达水平降低(P<0.01),P62和Bcl-2表达升高(P<0.01),p-PI3K/PI3K、p-Akt/Akt和p-mTOR/mTOR比值均显著升高(P<0.01)。结论:H2S可促进糖尿病大鼠皮肤创面愈合,其机制可能与激活PI3K/Akt/mTOR信号通路,抑制血管内皮细胞自噬及凋亡,促进血管生成有关。

AIM:To explore the effect and mechanism of hydrogen sulfide(H2S)on autophagy and angiogene-sis in skin wound of diabetic rats.METHODS:Among 36 healthy 8-week-old male Sprague-Dawley rats,12 rats were se-lected 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(56μmol/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 de-tected 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 cystathionineγ-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 deter-mined 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.Com-pared 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 au-tophagy and apoptosis,and promotion of angiogenesis in diabetic rats.