草氨酸钠通过抑制肺泡Ⅱ型上皮细胞衰老减轻小鼠矽肺纤维化

Oxamate alleviates silicotic fibrosis in mice by inhibiting senescence of alveolar type Ⅱ epithelial cells

[背景]肺泡Ⅱ型上皮细胞衰老是矽肺纤维化进展的重要驱动因素,草氨酸钠对肺泡Ⅱ型上皮细胞衰老的调节作用尚不清楚。[目的]探讨乳酸脱氢酶抑制剂草氨酸钠是否可通过抑制肺泡Ⅱ型上皮细胞衰老减轻小鼠矽肺纤维化。[方法]本研究分为体内实验和体外实验两部分。体内研究中将40只SPF级雄性C57BL/6J随机分为4组,每组10只,实验分组为对照组、矽肺模型组、草氨酸钠低剂量治疗组、草氨酸钠高剂量治疗组。采用一次性气管灌注SiO_(2)悬浊液50μL(100 mg·mL^(−1))制备矽肺小鼠模型;采用腹腔注射草氨酸钠100μL(225 mmol·L^(−1)和1125 mmol·L^(−1))制备草氨酸钠治疗模型。体外研究中采用SiO_(2)诱导MLE-12小鼠肺泡Ⅱ型上皮细胞,实验分组为①不同浓度SiO_(2)诱导组:对照组、50μg·mL^(−1)SiO_(2)组、100μg·mL^(−1)SiO_(2)组和200μg·mL^(−1)SiO_(2)组;②草氨酸钠治疗组:对照组、SiO_(2)组(100μg·mL^(−1))、草氨酸钠低剂量(25 mmol·L^(−1))治疗组和草氨酸钠高剂量(50 mmol·L^(−1))治疗组。采用苏木素-伊红(HE)染色观察肺组织病理学形态;天狼星红染色观察肺组织胶原蛋白沉积;采用免疫荧光染色观察表面活性蛋白C前体(Pro-SPC)和β-半乳糖苷酶(β-galactosidase)的阳性共表达;采用免疫荧光染色观察MLE-12细胞β-galactosidase的阳性表达;采用免疫印迹法检测I型胶原(CoL I)、纤维连接蛋白1(FN1)、己糖激酶2(HK2)、肌肉丙酮酸激酶同工酶2(PKM2)、乳酸脱氢酶A(LDHA)、磷酸化毛细血管扩张性共济失调(p-ATR)、细胞周期蛋白依靠性激酶抑制剂p21和p16的蛋白表达水平。[结果]与对照组相比较,矽肺模型组和SiO_(2)诱导的MLE-12细胞中,HK2、PKM2、LDHA、p-ATR、p21和p16的蛋白表达水平均上调(P<0.05)。体内研究显示,与对照组相比较,矽肺模型组中,矽结节面积,胶原蛋白沉积面积,β-galactosidase阳性细胞数占比,CoL I、FN1、LDHA、p-ATR、p21和p16的蛋白表达水平均上调(P<0.05);与矽肺模型组相比较,草氨酸钠治疗组中矽结节面积,胶原蛋白沉积面积,β-galactosidase阳性细胞数占比,CoL I、FN1、LDHA、p-ATR、p21和p16的蛋白表达水平均下调,且草氨酸钠高剂量治疗组效果优于草氨酸钠低剂量治疗组(P<0.05)。体外研究显示,与对照组相比较,SiO_(2)诱导组中,β-galactosidase阳性细胞数占比、p-ATR、p21和p16的蛋白表达水平均上调(P<0.05);与SiO_(2)组相比较,草氨酸钠治疗组中β-galactosidase阳性细胞数占比、LDHA、p-ATR、p21和p16的蛋白表达水平均下调,且草氨酸钠高剂量治疗组效果优于草氨酸钠低剂量治疗组(P<0.05)。[结论]乳酸脱氢酶抑制剂草氨酸钠可通过抑制肺泡Ⅱ型上皮细胞衰老以减轻小鼠矽肺纤维化。

[Background]The senescence of alveolar type Ⅱ epithelial cells is an important driving factor for the progression of silicotic fibrosis,and the regulatory effects of oxamate on the senescence of alveolar type Ⅱ epithelial cells is still unclear.[Objective]To explore whether lactate dehydrogenase inhibitor oxamate can alleviate silicotic fibrosis in mice by inhibiting senescence of alveolar type Ⅱ epithelial cells[Methods]This study was divided into two parts:in vivo experiments and in vitro experiments.In the first part,forty SPF C57BL/6J male mice were randomly divided into four groups with 10 in each group:control group,silicosis model group,low-dose oxamate treatment group,and high-dose oxamate treatment group.The silicotic mouse model was established by intratracheal instillation of 50μL SiO_(2)suspension(100 mg·mL^(−1)).The treatment models were prepared by intraperitoneal injection of 100μL oxamate(225 mmol·L^(−1)and 1125 mmol·L^(−1)).In the second part,induction of MLE-12 mouse alveolar type Ⅱ epithelial cells was conducted with SiO_(2).The in vitro experimental groups were①SiO_(2)induction groups:control group,50μg·mL^(−1)SiO_(2)group,100μg·mL^(−1)SiO_(2)group,and 200μg·mL^(−1)SiO_(2)group,and②oxamate treatment groups:control group,SiO_(2)group(100μg·mL^(−1)),low-dose oxamate(25 mmol·L^(−1))treatment group,and high-dose oxamate(50 mmol·L^(−1))treatment group.Pathological morphology of lung tissues was evaluated after hematoxylin-eosin(HE)staining;deposition of collagen in lung tissues was evaluated after sirius red staining;positive co-expression of prosurfactant protein C(Pro-SPC)andβ-galactosidase was detected by immunofluorescence staining;positive expression ofβ-galactosidase in MLE-12 cells was detected by immunofluorescence staining.The protein expression levels of collagen type I(CoL I),fibronectin1(FN1),hexokinase 2(HK2),pyruvate kinase isozyme type M2(PKM2),lactate dehydrogenase A(LDHA),p-ataxia telangiectasia and Rad3-related kinase(ATR),and cyclin-dependent kinase inhibitors p21,and p16 were detected by Western blotting.[Results]Compared with the control group,the protein expression levels of HK2,PKM2,LDHA,p-ATR,p21,and p16 were significantly upregulated in the silicosis model group and the SiO_(2)-induced MLE-12 cells(P<0.05).The in vivo studies showed that,compared with the control group,the silicon nodule area,the collagen deposition area,the proportion ofβ-galactosidase positive cells,and the protein expression levels of CoL I,FN1,LDHA,p-ATR,p21,and p16 were significantly upregulated in the silicosis model group(P<0.05).Compared with the silicosis model group,the oxamate treatment groups showed significant downregulation of the silicon nodule area,the collagen deposition area,the proportion ofβ-galactosidase positive cells,and the the CoL I,FN1,LDHA,p-ATR,p21,and p16 protein expression levels,and the high-dose oxamate treatment group showed a higher efficacy on these indicators than the low-dose oxamate treatment group(P<0.05).The in vitro studies showed that,compared with the control group,the proportion ofβ-galactosidase positive cells and the protein expression levels of p-ATR,p21,and p16 were significantly upregulated in the SiO_(2)-induced group(P<0.05).Compared with the SiO_(2)group,the proportion ofβ-galactosidase positive cells and the LDHA,p-ATR,p21 and p16 protein expression levels were significantly downregulated in the oxamate treatment groups,and the high-dose oxamate treatment group showed a higher efficacy on these indicators than the low-dose oxamate treatment group(P<0.05).[Conclusion]Lactate dehydrogenase inhibitor oxamate can alleviate silicotic fibrosis in mice by inhibiting the senescence of alveolar type Ⅱ epithelial cells.