褪黑素对链脲佐菌素诱导的糖尿病肺纤维化的作用及机制研究

Effect and mechanism of melatonin on streptozotocin-induced diabetic pulmonary fibrosis

目的探讨褪黑素对链脲佐菌素(streptozotocin,STZ)诱导的糖尿病肺纤维化的作用及其具体调控机制。方法将C57BL/6小鼠以随机数字表法随机分为对照组、STZ组、STZ+低剂量褪黑素(5 mg/kg)组、STZ+高剂量褪黑素(30 mg/kg)组,采用单次腹腔注射STZ(150 mg/kg)建立糖尿病肺纤维化小鼠模型,2周后血糖≥16.7 mmol/L为造模成功,随后给予褪黑素灌胃4周并喂养至16周处死取材。将人脐静脉内皮细胞分为对照组(葡萄糖浓度为5.5 mmol/L)、高糖组(葡萄糖浓度为33.3 mmol/L)、高糖+低剂量褪黑素(5μmol/L)组、高糖+高剂量褪黑素(20μmol/L)组,药物刺激结束后收集各组细胞用于后续检测。采用马松染色和免疫荧光染色观察肺纤维化病变,Western印迹法检测相关蛋白表达,采用转染沉默调节蛋白3(Sirtuin 3,Sirt3)siRNA的方法敲低Sirt3。结果与对照组相比,STZ组肺组织发生显著的纤维化病变,而STZ+低剂量褪黑素组、STZ+高剂量褪黑素组纤维化均较STZ组减少。同时,与对照组相比,STZ/高糖组内皮细胞标志物血小板-内皮细胞黏附分子(CD31)显著减少(P<0.001;P<0.001),间质纤维化标志物胶原蛋白3(Collagen 3)、波形蛋白(Vimentin)和α-平滑肌肌动蛋白(α-SMA)显著增加(P<0.001,P=0.035,P<0.001;P<0.001,P<0.001,P<0.001),而STZ/高糖+低剂量褪黑素组、STZ/高糖+高剂量褪黑素组在给予褪黑素治疗后上述趋势被部分逆转。此外,与对照组相比,STZ/高糖组Sirt3的蛋白表达显著减少(P<0.001;P<0.001),而STZ/高糖+低剂量褪黑素组、STZ/高糖+高剂量褪黑素组的Sirt3的蛋白表达较STZ/高糖组增加(P=0.047,P<0.001;P=0.048,P<0.001)。转染Sirt3 siRNA敲低Sirt3的表达后,与高糖+高剂量褪黑素组相比,高糖+高剂量褪黑素+Sirt3 siRNA组内皮细胞标志物CD31显著减少(P=0.026),间质纤维化标志物Collagen 3、Vimentin和α-SMA显著增加(P<0.001,P<0.001,P<0.001)。结论褪黑素通过激活Sirt3的表达抑制内皮间质转化,进而减轻STZ诱导的糖尿病小鼠肺纤维化。

ObjectiveTo explore the effects of melatonin on streptozotocin(STZ)-induced diabetic pulmonary fibrosis and regulatory mechanisms.MethodsC57BL/6 mice were randomly divided into the control group,STZ group,STZ+low-dose melatonin(5 mg/kg)group,STZ+high-dose melatonin(30 mg/kg)group using random number table,and a single intraperitoneal injection of STZ(150 mg/kg)was administered to establish a diabetic pulmonary fibrosis mouse model.Two weeks later,blood glucose levels≥16.7 mmol/L confirmed successful modeling.Subsequently,melatonin was administered orally for 4 weeks,and the mice were sacrificed at 16 weeks for tissue sampling.Human umbilical vein endothelial cells were divided into the control group(glucose concentration is 5.5 mmol/L),high glucose group(glucose concentration is 33.3 mmol/L),high glucose+low-dose melatonin(5μmol/L)group,high glucose+high-dose melatonin(20μmol/L)group,and cells in each group were collected for subsequent detection after drug stimulation.Masson staining and immunofluorescence staining were used to observe fibrotic lesions,Western blotting was used to detect the expression related proteins,and sirtuin 3(Sirt3)siRNA was transfected to knock down Sirt3.ResultsSignificant fibrotic lesions were observed in the lung tissue of the STZ group compared to the control group,however,the STZ+low-dose melatonin group and STZ+high-dose melatonin group showed reduced fibrosis compared to the STZ group.In addition,compared to the control group,the endothelial cell marker platelet endothelial cell adhesion molecule-1(CD31)was significantly decreased in the STZ/high glucose group(P<0.001;P<0.001),and the interstitial fibrosis markers collagen 3,Vimentin,andα-smooth muscle actin(α-SMA)were significantly increased(P<0.001,P=0.035,P<0.001;P<0.001,P<0.001,P<0.001),but these trends were partially reversed after melatonin treatment in the STZ/high glucose+low-dose melatonin group and the STZ/high glucose+high-dose melatonin group.Moreover,the protein expression of Sirt3 was significantly reduced in the STZ/high glucose group compared to the control group(P<0.001;P<0.001),while it was increased in the STZ/high glucose+low-dose melatonin and STZ/high glucose+high-dose melatonin groups compared to the STZ/high glucose group(P=0.047,P<0.001;P=0.048,P<0.001).After transfecting Sirt3 siRNA to knock down the expression of Sirt3,the endothelial cell marker CD31 was significantly reduced(P=0.026),and interstitial fibrosis markers collagen 3,Vimentin,andα-SMA were significantly increased in the high glucose+high-dose melatonin+Sirt3 siRNA group compared to the high glucose+high-dose melatonin group(P<0.001,P<0.001,P<0.001).ConclusionMelatonin inhibits endothelial-mesenchymal transition by activating Sirt3 expression,thereby alleviating pulmonary fibrosis in STZ-induced diabetic mice.