LncRNA HOTAIR Promotes LPS-Induced Inflammation and Apoptosis of Cardiomyocytes via Lin28-Mediated PDCD4 Stability.

Sepsis is one of the primary causes of death in intensive care units. Recently, increasing evidence has identified lncRNA HOTAIR is involved in septic cardiomyopathy. However, the potential mechanism underlying HOTAIR on septic cardiomyopathy is still unknown. H9C2 cells were treated with lipopolysaccharide (LPS) after transfection with sh-HOTAIR, sh-Lin28, pcDNA3.1-HOTAIR, and pcDNA3.1-PDCD4. qRT-PCR was used to examine the level of HOTAIR, Lin28, PDCD4, and sepsis-related inflammatory cytokines. Flow cytometric analysis was applied to detect cell apoptosis. The interaction between Lin28 and HOTAIR or PDCD4 was verified by RNA pull-down and RIP assay. HOTAIR levels were interfered by AAV9-sh-HOTAIR in LPS-induced septic cardiomyopathy mice. ELISA analysis was used to evaluate TNF-α, IL-6, and IL-1ß level. Western blot was used to detect the expression of LIN28 and PDCD4 in mouse cardiomyocytes. Echocardiography was used to evaluate the cardiac function. In our study, knockdown of HOTAIR inhibited LPS-induced inflammation and H9C2 cells apoptosis. HOTAIR promoted LPS-induced inflammatory response and apoptosis of H9C2 cells by enhancing PDCD4 stability. RNA pull-down and RIP assay exhibited that Lin28, a highly conserved RNA-binding protein, was combined with HOTAIR and PDCD4. The in vivo experiments verified that the HOTAIR knockdown alleviated the cardiac function injury and secretion of inflammatory factors caused by sepsis. In conclusion, our findings supported that the HOTAIR/Lin28/PDCD4 axis serves as a critical regulator of sepsis, which may open a new direction for the development of sepsis therapeutic.

Puerarin Alleviates Lipopolysaccharide-Induced Myocardial Fibrosis by Inhibiting PARP-1 to Prevent HMGB1-Mediated TLR4-NF-κB Signaling Pathway.

Myocardial fibrosis (MFs) is a crucial pathological process that results in cardiac failure in the development of multiple cardiovascular diseases. Puerarin could reportedly be used to treat a variety of cardiovascular diseases. However, the exact mechanism of puerarin on MFs was not clear enough. The separated primary cardiac fibroblasts (CFs) were induced by lipopolysaccharide (LPS) and treated with puerarin. The levels of TNF-α, IL-6, HMGB1, PARP-1, α-SMA, collagen-1, collagen-3, NF-κB pathways were examined by ELISA, immunofluorescence, RT-qPCR, western blot and immunohistochemistry assays. In addition, MFs rats' model was established using transverse aortic constriction (TAC), and the degree of fibrosis was certified by masson staining. We successfully separated primary CFs, and certified that LPS induction could upregulate the levels of PARP-1, HMGB1, inflammatory cytokines and fibrosis-related proteins (α-SMA, collagen-1 and collagen-3). In addition, we proved that puerarin could weaken MFs, and PARP-1 and HMGB1 expressions, which were induced by LPS in primary CFs. In terms of mechanism, HMGB1 expression could be promoted by PARP-1, and PARP-1 could attenuate the therapeutic effect of puerarin on LPS-induced MFs. Besides, PARP-1-HMGB1-NF-κB pathway was related to the protective effect of puerarin on MFs. In vivo, we also verified the protective efficacy of puerarin on MFs induced by TAC, and puerarin also regulated HMGB1-mediated TLR4-NF-κB signaling pathway. We demonstrated that puerarin could ameliorate MFs by downregulating PARP-1 to inhibit HMGB1-mediated TLR4-NF-κB signaling pathway in LPS-induced primary CFs and TAC-induced MFs rats' model.

Angiotensin II facilitates fibrogenic effect of TGF-ß1 through enhancing the down-regulation of BAMBI caused by LPS: a new pro-fibrotic mechanism of angiotensin II.

Angiotensin II has progressively been considered to play an important role in the development of liver fibrosis, although the mechanism isn't fully understood. The aim of this study was to investigate a possible pro-fibrotic mechanism, by which angiotensin II would enhance the pro-fibrotic effect of transforming growth factor beta 1 (TGF-ß1) through up-regulation of toll-like receptor 4 (TLR4) and enhancing down-regulation of TGF-ß1 inhibitory pseudo-receptor-BAMBI caused by LPS in hepatic stellate cells (HSCs). Firstly, the synergistic effects of angiotensin II, TGF-ß1 and LPS on collagen 1α production were confirmed in vitro by ELISA, in which angiotensin II, LPS and TGF-ß1 were treated sequentially, and in vivo by immunofluorescence, in the experiments single or multiple intra-peritoneally implanted osmotic mini-pumps administrating angiotensin II or LPS combined with intra-peritoneal injections of TGF-ß1 were used. We also found that only LPS and TGF-ß1 weren't enough to induce obvious fibrogenesis without angiotensin II. Secondly, to identify the reason of why angiotensin II is so important, the minute level of TLR4 in activated HSCs - T6 and primary quiescent HSCs of rat, up-regulation of TLR4 by angiotensin II and blockage by different angiotensin II receptor type 1 (AT1) blockers in HSCs were assayed by western blotting in vitro and immunofluorescence in vivo. Finally, BAMBI expression level, which is regulated by LPS-TLR4 pathway, was detected by qRT-PCR and results showed angiotensin II enhanced the down-regulation of BAMBI mRNA caused by LPS in vitro and in vivo, and TLR4 neutralization antibody blocked this interactive effect. These data demonstrated that angiotensin II enhances LPS-TLR4 pathway signaling and further down-regulates expression of BAMBI through up-regulation of TLR4, which results in facilitation of pro-fibrotic activity of TGF-ß1. Angiotensin II, LPS and TGF-ß1 act synergistically during hepatic fibrogenesis, showing crosstalks between angiotensin II-AT1, LPS-TLR4 and TGF-ß1-BAMBI signal pathways in rat HSCs.

Histones-mediated lymphocyte apoptosis during sepsis is dependent on p38 phosphorylation and mitochondrial permeability transition.

Lymphocyte apoptosis is one reason for immunoparalysis seen in sepsis, although the triggers are unknown. We hypothesized that molecules in plasma, which are up-regulated during sepsis, may be responsible for this. In this study, peripheral lymphocyte apoptosis caused by extracellular histones was confirmed both in mouse and human primary lymphocytes, in which histones induced lymphocyte apoptosis dose-dependently and time-dependently. To identify which intracellular signal pathways were activated, phosphorylation of various mitogen-activated protein kinases (MAPKs) were evaluated during this process, and p38 inhibitor (SB203580) was used to confirm the role of p38 in lymphocyte apoptosis induced by histones. To investigate the mitochondrial injury during these processes, we analyzed Bcl2 degradation and Rhodamine 123 to assess mitochondrial-membrane stability, via cyclosporin A as an inhibitor for mitochondrial permeability transition (MPT). Then, caspase 3 activation was also checked by western-blotting. We found that p38 phosphorylation, mitochondrial injury and caspase 3 activation occurred dose-dependently in histones-mediated lymphocyte apoptosis. We also observed that p38 inhibitor SB203580 decreased lymphocyte apoptotic ratio by 49% (P<0.05), and inhibition of MPT protected lymphocytes from apoptosis. Furthermore, to investigate whether histones are responsible for lymphocyte apoptosis, various concentrations of histone H4 neutralization antibodies were co-cultured with human primary lymphocytes and plasma from cecal ligation and puncture (CLP) mice or sham mice. The results showed that H4 neutralization antibody dose-dependently blocked lymphocyte apoptosis caused by septic plasma in vitro. These data demonstrate for the first time that extracellular histones, especially H4, play a vital role in lymphocyte apoptosis during sepsis which is dependent on p38 phosphorylation and mitochondrial permeability transition. Neutralizing H4 can inhibit lymphocyte apoptosis, indicating that it could be a potential target in clinical interventions for sepsis associated immunoparalysis.

[Differences of the regulation on the expression of mucin 1 induced by two single-strand RNA viruses, respiratory syncytial virus and influenza virus].

To investigate whether influenza virus (IFZ) could up-regulate the expression of mucin 1 (MUC1) which exists in epithelial cells of upper respiratory track to restrict the inflammation, as respiratory syncytial virus (RSV) does. Quantitative RT-PCR and Western Blot were performed to detect the expression level of MUC1 induced by two single-strand RNA viruses in A549 cell lines. HEp-2 and MDCK cells were used respectively to culture RSV and IFZ. At 24h post A549 cells infection with the same titer of RSV or IFZ, the total RNA was harvest, qRT-PCR was then performed to observe the expression level of MUC1 mRNA. Meanwhile, at 24 h and 48 h post A549 cells infection with the same titer of RSV or IFZ, the total protein and supernatant were collected respectively after cell lysis, Western Blot was then used to detect the expression level of MUC1. Results showed that RSV could up-regulate the expression of MUC1 in airway epithelial cells with a significant dose-effect correlation, whereas IFZ could not. This study firstly investigated the differences of the regulation on the expression of MUC1 induced by two single-strand RNA viruses, and demonstrated initially that the mechanism of IFZ self-limiting differed from RSV, which attributed to up-regulation of the expression level of MUC1.

Spironolactone lowers portal hypertension by inhibiting liver fibrosis, ROCK-2 activity and activating NO/PKG pathway in the bile-duct-ligated rat.

OBJECTIVE: Aldosterone, one of the main peptides in renin angiotensin aldosterone system (RAAS), has been suggested to mediate liver fibrosis and portal hypertension. Spironolactone, an aldosterone antagonist, has beneficial effect on hyperdynamic circulation in clinical practice. However, the mechanisms remain unclear. The present study aimed to investigate the role of spionolactone on liver cirrhosis and portal hypertension. METHODS: Liver cirrhosis was induced by bile duct ligation (BDL). Spironolactone was administered orally (20 mg/kg/d) after bile duct ligation was performed. Liver fibrosis was assessed by histology, Masson's trichrome staining, and the measurement of hydroxyproline and type I collagen content. The activation of HSC was determined by analysis of alpha smooth muscle actin (α-SMA) expression. Protein expressions and protein phosphorylation were determined by immunohistochemical staining and Western blot analysis, Messenger RNA levels by quantitative real time polymerase chain reaction (Q-PCR). Portal pressure and intrahepatic resistance were examined in vivo. RESULTS: Treatment with spironolactone significantly lowered portal pressure. This was associated with attenuation of liver fibrosis, intrahepatic resistance and inhibition of HSC activation. In BDL rat liver, spironolactone suppressed up-regulation of proinflammatory cytokines (TNFα and IL-6). Additionally, spironolactone significantly decreased ROCK-2 activity without affecting expression of RhoA and Ras. Moreover, spironolactone markedly increased the levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS and the activity of NO effector-protein kinase G (PKG) in the liver. CONCLUSION: Spironolactone lowers portal hypertension by improvement of liver fibrosis and inhibition of intrahepatic vasoconstriction via down-regulating ROCK-2 activity and activating NO/PKG pathway. Thus, early spironolactone therapy might be the optional therapy in cirrhosis and portal hypertension.