Sirtuin 6 regulates macrophage polarization to alleviate sepsis-induced acute respiratory distress syndrome via dual mechanisms dependent on and independent of autophagy.

BACKGROUND AIMS: Sepsis-induced acute respiratory distress syndrome (ARDS) can be mediated by an imbalance in macrophage polarization; however, the underlying mechanisms remain poorly understood. This study aimed to investigate the modulatory role of sirtuin 6 (SIRT6) in macrophage polarization during sepsis-induced ARDS. METHODS: A mouse ARDS model was established using cecal ligation and puncture. Isolated alveolar macrophages (AMs) and lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDMs) were adopted as in vitro models. Macrophage polarization was evaluated by measuring M1 and M2 macrophage percentages via flow cytometry and expression of specific markers. The expression of microtubule-associated light chain protein 3I/II and beclin-1 was detected for assessing macrophage autophagy. Binding between specificity protein 1 (SP1) and the target gene promoter was evaluated using a chromatin immunoprecipitation assay. RNA expression was analyzed by quantitative reverse transcription polymerase chain reaction and western blotting. RESULTS: Treatment with the SIRT6 activator UBCS039 significantly alleviated lung injury in the mouse ARDS model and enhanced autophagy and M2 polarization in isolated AMs. M2 polarization and autophagy in LPS-challenged BMDMs were also effectively promoted by UBCS039 treatment or SIRT6 overexpression. An adenosine monophosphate-activated protein kinase inhibitor (Compound C) or autophagy inhibitor (3-methyladenine) partially abrogated M2 polarization mediated by SIRT6 overexpression upon LPS exposure. SIRT6 induced autophagy and M2 polarization of BMDMs partially via its deacetylase activity. SIRT6 inhibited mammalian target of rapamycin transcription by modulating SP1 to promote BMDM M2 polarization, which was independent of autophagy. CONCLUSIONS: SIRT6 promotes M2 polarization of macrophages to alleviate sepsis-induced ARDS in an autophagy-dependent and -independent manner.

Ginsenoside Rg1 Regulates SIRT1 to Ameliorate Sepsis-Induced Lung Inflammation and Injury via Inhibiting Endoplasmic Reticulum Stress and Inflammation.

OBJECTIVES: To investigate the protective effect of ginsenoside Rg1 on relieving sepsis-induced lung inflammation and injury in vivo and in vitro. METHODS: Cultured human pulmonary epithelial cell line A549 was challenged with LPS to induce cell injury, and CLP mouse model was generated to mimic clinical condition of systemic sepsis. Rg1 was applied to cells or animals at indicated dosage. Apoptosis of cultured cells was quantified by flow cytometry, along with ELISA for inflammatory cytokines in supernatant. For septic mice, lung tissue pathology was examined, plus ELISA assay for serum cytokines. Western blotting was used to examine the activation of inflammatory pathways and ER stress marker proteins in both cells and mouse lung tissues. Reactive oxygen species (ROS) level was quantified by DCFDA kit. RESULTS: Ginsenoside Rg1 treatment remarkably suppressed apoptosis rate of LPS-induced A549 cells, relieved mouse lung tissue damage, and elevated survival rate. Rg1 treatment also rescued cells from LPS-induced intracellular ROS. In both A549 cells and mouse lung tissues, further study showed that Rg1 perfusion significantly suppressed the secretion of inflammatory cytokines including tumor necrosis factor- (TNF-) alpha and interleukin- (IL-) 6 and relieved cells from ER stress as supported by decreased expression of marker proteins via upregulating sirtuin 1 (SIRT1). CONCLUSION: Our results showed that ginsenoside Rg1 treatment effectively relieved sepsis-induced lung injury in vitro and in vivo, mainly via upregulating SIRT1 to relieve ER stress and inflammation. These findings provide new insights for unrevealing potential candidate for severe sepsis accompanied with lung injury.

lncRNA PVT1 modulates NLRP3­mediated pyroptosis in septic acute kidney injury by targeting miR­20a­5p.

Acute kidney injury (AKI) is the most common complication of sepsis. The current incidence of sepsis is high (0.3% of total population) worldwide, and septic AKI may cause death in patients. Long non­coding (lnc)RNAs serve important roles in the pathogenesis of AKI. Therefore, the present study investigated the mechanism underlying lncRNA plasmacytoma variant translocation 1 (PVT1)­mediated regulation of pyroptosis in septic AKI. Septic kidney injury was induced in mice using the caecal ligation and puncture method, and lipopolysaccharide (LPS)­induced HK­2 cell models were also established. Haematoxylin­eosin staining was performed to assess pathological alterations of kidney tissues in the mice. The levels of IL­1ß, IL­18 and lactate dehydrogenase were determined by conducting ELISAs. Reverse transcription­quantitative PCR was used to detect the expression levels of PVT1 and microRNA (miR)­20a­5p. To assess pyroptosis, the protein expression levels of nucleotide­binding oligomerization domain­like receptor protein 3 (NLRP3), IL­1ß, IL­18, apoptosis­associated speck­like protein containing a CARD and cleaved caspase­1 were measured via western blotting. Flow cytometry was performed to assess the rate of cell pyroptosis. Dual luciferase reporter assays were used to assess the binding relationships of PVT1/miR­20a­5p and miR­20a­5p/NLRP3. PVT1 expression was significantly increased, whereas miR­20a­5p expression was significantly decreased in sepsis model mice and LPS­induced HK­2 cells compared with sham mice and control HK­2 cells, respectively. PVT1 knockdown significantly suppressed cell pyroptosis and downregulated the expression of inflammatory factors in LPS­induced HK­2 cells. The results also indicated that PVT1 served as a sponge of miR­20a­5p, and miR­20a­5p directly targeted NLRP3. miR­20a­5p knockdown significantly promoted LPS­induced cell pyroptosis. Moreover, PVT1 knockdown inhibited LPS­induced cell pyroptosis by targeting the miR­20a­5p/NLRP3 signalling pathway. The results of the present study suggested that PVT1 modulated NLRP3­mediated pyroptosis in septic AKI by targeting miR­20a­5p, which might suggest significant potential therapeutic targets for septic AKI.

Ginsenoside Rg3 alleviates septic liver injury by regulating the lncRNA TUG1/miR-200c-3p/SIRT1 axis.

BACKGROUND: Studies have shown that ginsenoside R3 (Rg3) plays a protective role in sepsis-induced organ injuries and mitochondrial dysfunction. Long noncoding RNA (lncRNA) taurine-upregulated gene 1 (TUG1) is regarded as a regulator in sepsis. However, the association between TUG1 and Rg3 remains elusive. METHODS: A sepsis mouse model was established by caecal ligation and puncture (CLP), and liver injury was induced by haematoxylin-eosin (H&E) staining. Lipopolysaccharide (LPS) was used to induce hepatocyte damage. The expression levels of TUG1, microRNA (miR)-200a-3p, and silencing information regulator 1 (SIRT1) were examined by quantitative real-time polymerase chain reaction (qRT-PCR) assays. Cell viability was monitored using the Cell Counting Kit-8 (CCK-8) assay. MitoSOX Red staining and CBIC2 (JC-1) dye were employed to detect mitochondrial reactive oxygen species (ROS) and mitochondrial transmembrane potential (MTP) levels, respectively. The interaction between miR-200a-3p and TUG1 or SIRT1 was confirmed via dual-luciferase reporter or RNA immunoprecipitation (RIP) assay. RESULTS: Rg3 upregulated TUG1 expression in liver tissues of CLP mice and LPS-induced hepatocytes. Rg3 could activate autophagy to improve mitochondrial dysfunction in LPS-treated hepatocytes, which was partially reversed by TUG1 depletion or miR-200a-3p overexpression. Importantly, TUG1 targeted miR-200a-3p to activate the SIRT1/AMP-activated protein kinase (AMPK) pathway in LPS-treated hepatocytes. Moreover, gain of TUG1 ameliorated mitochondrial dysfunction in LPS-treated hepatocytes by sequestering miR-200a-3p. CONCLUSION: Our study revealed that Rg3 increased TUG1 expression and reduced miR-200a-3p expression to stimulate the SIRT1/AMPK pathway, thereby enhancing autophagy to improve sepsis-induced liver injury and mitochondrial dysfunction.

ROCK1 regulates sepsis-induced acute kidney injury via TLR2-mediated endoplasmic reticulum stress/pyroptosis axis.

BACKGROUND: It has been reported that ROCK1 participates in the progression of multiple diseases, including septic intestinal barrier, cardiac dysfunction and acute lung injury. However, its regulatory role and specific mechanism in sepsis-induced acute kidney injury (AKI) remain unclear. METHODS: Cecal ligation puncture (CLP) was conducted to establish sepsis mouse model, and in vitro model was achieved by lipopolysaccharide (LPS) stimulation. Genes expression was evaluated by qRT-PCR, western blot or ELISA was conducted to assess the levels of proteins. Hoechst staining was performed to evaluate cell pyroptosis. LDH activity assay was detected to assess cytotoxicity. Immunohistochemistry was conducted to detect Ly-6G expression and neutrophils distribution in kidney tissues of mice. H&E and TUNEL staining were carried to evaluate kidney injury of mice. RESULTS: Our findings illuminated that ROCK1 was highly expressed in sepsis-induced AKI, and ROCK1 knockdown inhibited NLRP3-mediated cell pyroptosis in LPS-induced HK-2 cells. Moreover, ROCK1 modulated HK-2 cell pyroptosis by regulating endoplasmic reticulum stress (ERS). TLR2 inhibitor could suppress ERS mediated cell pyroptosis under LPS treatment. Further, TLR2 activator partially reversed the effects of ROCK1 inhibition on ERS mediated pyroptosis in LPS-treated HK-2 cells and CLP mice. CONCLUSION: In conclusion, ROCK1 may regulate sepsis-induced AKI via TLR2-mediated ERS/pyroptosis axis. Our data demonstrated the role and underlying mechanism of ROCK1 in septic AKI, providing theoretical basis for sepsis-induced AKI treatment.