Knockdown of lncRNA LUCAT1 attenuates sepsis­induced myocardial cell injury by sponging miR-642a.

The heart is one of the most common organs involved in sepsis-induced organ dysfunction and about 50% septic patients complicated with myocardial injury. So far, the molecular mechanisms underlying sepsis-induced cardiac damage remain unclear. In this study we aimed to evaluate the effect of miR-642a on sepsis-induced cardiac injury in vitro and explore the possible lncRNA-microRNA mechanism. We first downloaded GSE101639 to identify differentially expressed genes (DEGs) in sepsis. The expression of miR-642a in LPS-induced H9C2 cells was detected by qRT-PCR. MTT assay, cell migration, flow cytometry analysis, ELISA, qRT-PCR and Western blotting analysis were applied to evaluating the effect of miR-642a mimic on LPS-induced H9C2 cells. The bioinformatics analysis and the rescue experiment were devoted to the underlying mechanism. The results showed miR-642a expression was decreased in septic patients and LPS-induced H9C2 cells. Besides, MiR-642a mimic promoted cell viability and migration, inhibited cell apoptosis of LPS-induced H9C2 cells. Bioinformatics analysis showed miR-642a directly targets with 3'-UTR of ROCK1. Moreover, LUCAT1 regulated ROCK1 expression act as a competing endogenous RNA (ceRNA) for miR-642a. Our data demonstrated that lncRNA LUCAT1 could function via sponging miR-642a to regulate ROCK1 expression in LPS-induced H9C2 cells. And knockdown of lncRNA LUCAT1 could suppress LPS-induced cardiac injury in vitro.

[Effects of Xuebijing injection on protein C and tumor necrosis factor-alpha mRNA in rats with sepsis].

OBJECTIVE: To investigate the effects of the integrated traditional Chinese medicine Xuebijing injection on protein C (PC) and tumor necrosis factor-alpha (TNF-alpha) mRNA in rats with sepsis. METHODS: Sepsis was induced in Wistar rats by cecal ligation and puncture (CLP). Ninety-six healthy animals were randomly divided into four groups: normal group, sham-operation group, CLP model group, and Xuebijing-treated group. The two latter groups were divided into 2, 8, 24, 48, and 72-hour subgroups with 8 rats in each subgroup. Platelet count of blood obtained from abdominal aorta was determined and tissue samples from liver and lungs were collected to measure tissue PC and TNF-alpha mRNA expression. RESULTS: PC gene expression levels in lung tissues were significantly lowered (all P<0.01), but they were dramatically raised by Xuebijing injection during 8-72 hours post-CLP (all P<0.01). Compared with normal group, TNF-alpha mRNA levels in liver and lungs were significantly elevated at 2 hours post-CLP (P<0.05 or P<0.01). However, treatment with Xuebijing injection markedly reduced TNF-alpha mRNA both in liver and lungs at 2-24 hours (P<0.05 or P<0.01). In CLP group, blood platelet count was significantly decreased to certain extent at different intervals within 8-72 hours, and it was markedly elevated in the Xuebijing-treated group (P<0.05 or P<0.01). CONCLUSION: The current study suggests that Xuebijing injection could exert preventing effect on the development of severe sepsis by suppressing PC and TNF-alpha mRNA.

[Effects of Xuebijing injection on thrombomodulin and endothelial cell protein C receptor in septic rats].

OBJECTIVE: To investigate effects of the integrated traditional Chinese medicine Xuebijing injection on thrombomodulin (TM) and endothelial cell protein C receptor (EPCR) in septic rats. METHODS: Ninety-six healthy Wistar rats were randomly divided into four groups: control group, sham operation group, cecal ligation and puncture (CLP) model group, and Xuebijing-treated group. Sepsis was reproduced by CLP. The two latter groups were divided into five subgroups of 2, 8, 24, 48 and 72-hour with 8 rats in each subgroup. Tissue samples from liver and lung were collected to determine tissue TM and EPCR mRNA expression. RESULTS: TM and EPCR mRNA expressions were observed in liver and lung in control group and sham operation group, while with no significant differences at 2 hours post-CLP (both P>0.05). TM and EPCR gene expression levels in tissues were significantly increased to certain extent at 8-48 hours (all P<0.01), and were dramatically decreased following Xuebijing injection at 72 hours post-CLP (both P>0.05). Also, treatment with Xuebijing injection markedly decreased TM and EPCR mRNA levels to certain extents at 8 and 24 hours, and markedly increased at 48 and 72 hours compared with those of model group. CONCLUSION: These data suggest that Xuebijing injection could raise TM and EPCR mRNA expression, thereby it might be effective in prevention of development of severe sepsis.

[Treatment effects of antithrombin-III on coagulation abnormalities in rats with endotoxaemia].

OBJECTIVE: To study the treatment effects of antithrombin-III (AT-III) on coagulation abnormalities in rats with endotoxaemia. METHODS: Twenty-four Wistar rats were randomly divided into control group, coagulation abnormality group and AT-III group (each n=8). Endotoxaemia coagulopathy model was reproduced by intravenous injection of lipopolysaccharide (LPS) in two doses of 1.4 ml/kg (100 microg) and 2.8 ml/kg (200 microg) 12 hours apart. In the AT-III group, AT-III 25 U/kg was given intravenously 1 hour after second injection of LPS. The changes in blood platelet (PLT) count, activated partial thromboplastin time (APTT), prothrombin time (PT), D-dimer (DD), AT-III activity, fibrinogen (FI), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were determined 3 hours later. RESULTS: Levels of PLT, FI and AT-III in coagulation abnormality group were lowered compared with control group (all P<0.01), while APTT, DD, PT, ALT, AST, ALP and LDH were increased (all P<0.01). All indexes were significantly improved in AT-III group, the values were close to those of normal control group (all P>0.05), and the differences were significant when compared with those of coagulation abnormality group (all P<0.01). Pathological changes of the lung, kidney and liver tissues were lighter in AT-III group than those of coagulation abnormality group. CONCLUSION: These findings indicate that AT-III can be used to treat disseminated intravascular coagulation (DIC) in rats with endotoxaemia. Preventive use of AT-III in rats with endotoxaemia is therapeutically effective.

[Reproduction of a rat model of disseminated intravascular coagulation accompanied by multiple organ injuries].

OBJECTIVE: To reproduce a rat model of disseminated intravascular coagulation (DIC) accompanied by multiple organ dysfunction syndrome (MODS) induced by endotoxin. METHODS: Twenty-four healthy Wistar rats were randomly divided into four groups: control group, low dosage lipopolysaccharide (LPS) group, middle dosage LPS group, and high dosage LPS group (each n=6). Rats of each group were given different dosages of normal saline (1.4 ml/kg, 2.8 ml/kg), low dosages LPS [1.4 mg/kg (56 microg/kg), 2.8 ml/kg (112 microg/kg)], middle dosages LPS [1.4 mg/kg(98 microg/kg), 2.8 ml/kg (196 microg/kg)] and high dosages LPS [1.4 ml/kg (196 microg/kg), 2.8 ml/kg (392 microg/kg)] respectively twice 12 hours apart through femoral vein intubation injection. Blood platelet (PLT) count, coagulation function, D-dimer, fibrinogen (Fbg), antithrombin III (AT-III) blood glucose (Glu), biochemistry indexes including aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were determined before and after LPS challenge, and histopathologic changes of lung, liver and kidney were observed 4 hours after the second injection. RESULTS: The rats in high dosage LPS group died 4 hours later, and the rats in low and middle dosage LPS groups survived after double LPS challenge. The results of blood PLT, D-dimer, Fbg, Glu, AST, ALT, ALP, LDH, coagulation function of activated partial thromboplastin time, prothrombin time and level of anti-thrombin III in middle dosage LPS group were significantly different compared with those of control group (P<0.05 or P<0.01). Obvious changes in histopathology were found in major organs such as lung, kidney and liver. CONCLUSION: Double intravenous LPS challenge (98 microg/kg and 196 microg/kg) in rats can reproduce a rat model with DIC accompanied by multiple organ injuries.

[Effects of antithrombin-III on cytokines in rats with multiple organ dysfunction syndrome].

OBJECTIVE: To observe the effect of the antithrombin-III (AT-III) on cytokine levels in multiple organ dysfunction syndrome (MODS) in rats. METHODS: Seventy Wistar rats were randomly divided into three groups: normal control group, MODS group, and AT-III treatment group. The rats in MODS group and the treatment group were given lipopolysaccharide (LPS) two times to replicate systemic inflammatory response syndrome (SIRS)/MODS model. One hour after the injections of LPS, the rats in the treatment group were given AT-III (25 U/kg, 0.5 ml/100 g) intravenously. Four hours after treatment, blood samples were collected in rats. The tissue samples were collected and preserved in formalin. By use of radioimmunoassay and histopathology, the effect of the AT-III was observed in rats with SIRS/MODS. RESULTS: Endotoxin (ET), interleukin-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF-alpha) levels were all increased significantly in MODS animals (all P<0.01), and they were decreased after AT-III treatment (all P<0.01), though still higher than those of the normal controls (all P<0.01). The pathologic changes in lung, kidney and liver in the treatment group were milder than in the MODS group. CONCLUSION: These data suggest that AT-III could inhibit systemic inflammatory response and it might be used in the treatment of SIRS/MODS.