摘要
为探索miR-34b-5p对脓毒症急性肺损伤(acute lung injury,ALI)大鼠的保护作用及对磷脂酶和张力蛋白同源物(phosphatase and tensin homolog,PTEN)/Akt/mTOR通路的影响,80只无特定病原体级雄性SD大鼠被随机分为假手术组、模型组、空载组和miR-34b-5p组,每组20只。假手术组大鼠仅开腹,其余大鼠采用盲肠结扎穿孔法制备脓毒症模型。模型建立后,空载组和miR-34b-5p组大鼠分别给予尾静脉注射空载体和miR-34b-5p过表达质粒,模型组、假手术组大鼠注射等量生理盐水。注射3 d后,用ELISA测定大鼠外周血IL-1β、IL-6水平,计算大鼠肺组织湿重/干重(wet weight/dry weight,W/D)比值,H-E染色观察大鼠肺组织病理改变,TUNEL法检测肺组织细胞凋亡率,qRT-PCR检测肺组织miR-34b-5p、IL-1β、IL-6 mRNA水平,Western blotting检测PTEN、p-Akt、p-mTOR、Akt、mTOR、B细胞淋巴瘤2(B cell lymphoma 2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)及Caspase-3蛋白表达。结果显示,假手术组大鼠肺泡正常,模型组、空载组大鼠肺泡塌陷,肺泡壁增厚,血管充血,肺间质充血、水肿,炎症细胞浸润,miR-34b-5p组大鼠肺组织炎症细胞浸润减少,肺泡损伤显著改善。与假手术组比较,模型组大鼠肺组织IL-1β、IL-6蛋白及mRNA水平,组织凋亡细胞数量,Bax、Caspase-3蛋白表达水平,PTEN蛋白表达水平及W/D比值显著升高(P<0.01),而miR-34b-5p表达水平、Bcl-2蛋白表达水平、p-Akt/Akt比值、p-mTOR/mTOR比值显著降低(P<0.01)。与空载组比较,miR-34b-5p组大鼠肺组织miR-34b-5p表达水平、Bcl-2蛋白表达水平、p-Akt/Akt及p-mTOR/mTOR比值显著升高(P<0.01),IL-1β、IL-6蛋白及mRNA水平,组织凋亡细胞数量,Bax、Caspase-3蛋白表达水平,PTEN蛋白表达水平及W/D比值显著降低(P<0.01)。该研究提示,miR-34b-5p可通过PTEN/Akt/mTOR通路减轻脓毒症ALI大鼠的肺部炎症和细胞凋亡。
To explore the protective effect of miR-34b-5p on sepsis induced acute lung injury(ALI)in rats and its impact on the phosphatase and tensin homolog(PTEN)/Akt/mTOR pathway,80 specific pathogen free grade male SD rats were randomly divided into the sham group,the model group,the empty vector group,and the miR-34b-5p group,with 20 rats in each group.The rats in the sham group only underwent laparotomy,while the remaining rats were subjected to cecal ligation and perfora-tion to establish a sepsis model.After the establishment of the model,rats in the empty vector group and the miR-34b-5p group were injected with empty vectors and miR-34b-5p overexpression plasmids through the tail vein,respectively.Rats in the model group and the sham group were injected with equal amount of physiological saline.Three days after injection,ELISA was used to determine the IL-1p and IL-6 levels in rat peripheral blood.The wet weight/dry weight(W/D)ratio of rat lung tissue was calculated and the pathological changes of rat lung tissue was observed by H-E staining.The apoptosis of lung histiocyte cells was detected by TUNEL method.miR-34b-5p,IL-1βand IL-6 mRNA levels in lung tissue were detected by qRT-PCR.Western blotting was used to detect the protein levels of PTEN,p-Akt,P-mTOR,Akt,mTOR,B cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),and Caspase-3.The results showed that the alveoli in the sham group appeared normal,while those of the model group and the empty vector group were collapsed,along with thickened alveolar walls,vascular congestion,pulmonary interstitial congestion,edema,and inflammatory cell infiltration.The infiltration of inflammatory cells in the lung tissue of rats in the miR-34b-5p group was reduced,and alveolar injury was significantly improved.Compared to those of the sham group,the expressions of IL-1βand IL-6 at both protein and mRNA levels,the number of apoptotic cells,levels of Bax,Caspase-3 and PTEN as well as W/D ratio were significantly increased in the model group(P<0.01),while the levels of miR-34b-5p,Bcl-2 protein,p-Akt/Akt ratio,and p-mTOR/mTOR ratio were significantly decreased(P<0.01).Compared to those of the empty vector group,the miR-34b-5p expression,Bcl-2 protein level,p-Akt/Akt and p-mTOR/mTOR ratio in the lung tissue of the miR-34b-5p group were significantly increased(P<0.01),while the IL-6,IL-1βprotein and mRNA levels,the number of apoptotic cells,the Bax,PTEN,Caspase-3 protein levels,and W/D ratio were significantly decreased(P<0.01).This study suggests that miR-34b-5p alleviates pulmonary inflammation and cell apoptosis of ALI in sepsis rats through the PTEN/Akt/mTOR pathway.
作者
张红玉
李新
吴勤奋
龙小艳
ZHANG Hongyu;LI Xin;WU Qinfen;LONG Xiaoyan(Department of ICU,the Second Affiliated Hospital of Xinjiang Medical University,Urumqi 830063,China)
出处
《现代免疫学》
2025年第4期402-408,共7页
Current Immunology
基金
省部共建中亚高发病成因与防治国家重点实验室(SKL-HIDCA-2023-EF5)
新疆维吾尔自治区自然科学基金项目(2021D01C361)。
