[The role of miR-135b-5p in inhibiting mice acute lung injury (ALI) induced by sepsis and its mechanism].

To investigate the expression status of miR-135b-5p in the sepsis induced acute lung injury (ALI) mice and its effects on inflammatory responses and cell pyroptosis in mice pulmonary tissues. The cecal ligation puncture (CLP) method was employed to construct sepsis-induced ALI mice models. The C57BL/6 mice were randomly divided into 6 groups with 8 mice in each group. The sepsis mouse models were constructed by performing CLP surgery: mice were anesthetized by intraperitoneal injection of 0.1 mg/kg barbital, the abdomen was cut longitudinally to expose the cecum, the cecum was ligated and perforated with syringe needle, the wound was sutured after extruding part of the intestinal contents. The sham operation group (Sham group) did not undergo any treatment and suture wounds after laparotomy, and no CLP operation was performed. The treatment groups were divided into CLP+NC mimic group, CLP+ miR-135b-5p mimic group, CLP+NC mimic+ Empty vector group, CLP+GSDMD group, and CLP+ miR-135b-5p mimic+GSDMD group. One week before CLP surgery, mice in the treatment group were injected subcutaneously with 200 μ L NC mimic (200 nmol/L), miR-135b-5p mimic (200 nmol/L), Empty vector (100 nmol/L), GSDMD Vector (100 nmol/L), and miR-135b-5p mimic (200 nmol/L), once a day for a week. The euthanasia was performed 24 h after operation by carbon dioxide asphyxiation. The qRT-PCR was utilized to determine miR-135b-5p and GSDMD expressions;HE staing assay was performed to observe the pathological changes of pulmonary tissues. The mice right lung tissues were flushed with 5 ml saline for 3 times, and each flush lasted for 3~5 min to collect the BALF, and the levels of GSDMD, IL-1β and IL-18 in BALF were determined by ELISA. The protein levels of NLRP3, caspase 1 and cleaved GSDMD in mice lung tissues were examined by immunoblotting analysis; Dual-luciferase reporter gene system assay was employed to validate the targeting relationship of miR-135b-5p and GSDMD. Compared with the control group mice, there were a large number of inflammatory cell infiltration, alveolar damage, interstitial edema and alveolar collapse in the lung tissues of the CLP group mice (P<0.01), and the expression levels of the pyroptosis-associated proteins (NLRP3, caspase-1 and GSDMD) were all increased, while miR-135b-5p was down-regulated in the CLP group (P<0.01). Further experiments confirmed that overexpression of miR-135b-5p significantly reduced CLP-induced pyroptotic cell death in mice lung tissues (P<0.01), and dual-luciferase reporter gene system assay confirmed that miR-135b-5p targeted GSDMD for its degradation. Moreover, the rescuing experiments validated that up-regulation of GSDMD abrogated the inhibition effects of miR-135b-5p overexpression on cell pyroptosis in CLP mice lung tissues (P<0.01). Consistently, we verified that miR-135b-5p also suppressed the expression levels of IL-1β and IL-18 in mice BALF via degrading GSDMD (P<0.05). Overexpression of miR-135b-5p attenuated sepsis-induced ALI by inhibiting GSDMD-mediated pyroptotic cell death, and this study provided potential therapeutic target and theoretical foundation for sepsis-induced ALI treatment.