Sharmila Adhikari

Hydrogen Sulfide Up-Regulates Substance P in Polymicrobial Sepsis-Associated Lung Injury

Hydrogen sulfide (H2S) has been shown to induce the activation of neurogenic inflammation especially in normal airways and urinary bladder. However, whether endogenous H2S would regulate sepsis-associated lung inflammation via substance P (SP) and its receptors remains unknown. Therefore, the aim of the study was to investigate the effect of H2S on the pulmonary level of SP in cecal ligation and puncture (CLP)-induced sepsis and its relevance to lung injury. Male Swiss mice or male preprotachykinin-A gene knockout (PPT-A−/−) mice and their wild-type (PPT-A+/+) mice were subjected to CLP-induced sepsis. DL-propargylglycine (50 mg/kg i.p.), an inhibitor of H2S formation was administered either 1 h before or 1 h after the induction of sepsis, while NaHS, an H2S donor, was given at the same time as CLP. L703606, an inhibitor of the neurokinin-1 receptor was given 30 min before CLP. DL-propargylglycine pretreatment or posttreatment significantly decreased the PPT-A gene expression and the production of SP in lung whereas administration of NaHS resulted in a further rise in the pulmonary level of SP in sepsis. PPT-A gene deletion and pretreatment with L703606 prevented H2S from aggravating lung inflammation. In addition, septic mice genetically deficient in PPT-A gene or pretreated with L703606 did not exhibit further increase in lung permeability after injection of NaHS. The present findings show for the first time that in sepsis, H2S up-regulates the generation of SP, which contributes to lung inflammation and lung injury mainly via activation of the neurokinin-1 receptor.

Effect of mitogen-activated protein kinases on chemokine synthesis induced by substance P in mouse pancreatic acinar cells.

Substance P, acting via its neurokinin 1 receptor (NK1 R), plays an important role in mediating a variety of inflammatory processes. Its interaction with chemokines is known to play a crucial role in the pathogenesis of acute pancreatitis. In pancreatic acinar cells, substance P stimulates the release of NFκB‐driven chemokines. However, the signal transduction pathways by which substance P‐NK1 R interaction induces chemokine production are still unclear. To that end, we went on to examine the participation of mitogen‐activated protein kinases (MAPKs) in substance P‐induced synthesis of pro‐inflammatory chemokines, monocyte chemoanractant protein‐1 (MCP‐I), macrophage inflammatory protein‐lα (MIP‐lα) and macrophage inflammatory protein‐2 (MIP‐2), in pancreatic acini. In this study, we observed a time‐dependent activation of ERK1/2, c‐Jun N‐terminal kinase (JNK), NFκB and activator protein‐1 (AP‐1) when pancreatic acini were stimulated with substance P. Moreover, substance P‐induced ERK 1/2, JNK, NFκB and AP‐1 activation as well as chemokine synthesis were blocked by pre‐treatment with either extracellular signal‐regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor. In addition, substance P‐induced activation of ERK 112, JNK, NFκB and AP‐1‐driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells. Taken together, these results suggest that substance P‐NK1 R induced chemokine production depends on the activation of MAPKs‐mediated NFκB and AP‐1 signalling pathways in mouse pancreatic acini.

Expression of Nitric Oxide Synthase Isoforms and Nitric Oxide Production in Acute Pancreatitis and Associated Lung Injury

Background/Aims: The role of nitric oxide (NO) has been increasingly implicated in the pathophysiology of acute pancreatitis (AP). Studies have shown increased NO production in AP although not all are agreeable on whether NO is beneficial or detrimental in AP. This study aims to profile NO production and NO synthase (NOS) expression in the pancreas and lungs in the progression of AP in mice to gain insights to the role played by different NOS isoforms. Methods: AP was induced in mice by hourly administration of cerulein. NO production was determined by measuring the total nitrite and nitrate (NOx) content while NOS expression was measured by Western blot. Results: Pancreatic NO production increased sharply and was sustained throughout AP. iNOS expression was greatly increased while eNOS was downregulated at the later stages. In the lungs, there was an unexpected early increase in the constitutive NOS expression; however iNOS was also significantly overexpressed at the later time point along with a significant increase in NO. Acinar cells were found to overproduce NO in response to cerulein hyperstimulation with iNOS again being the major contributor. Conclusion: These data show that NO production and NOS expression are differentially regulated temporally and in magnitude in the pancreas and lungs in response to cerulein hyperstimulation which suggests differing roles for each NOS isoform.

H2S‐induced pancreatic acinar cell apoptosis is mediated via JNK and p38 MAP kinase

Treatment of pancreatic acinar cells by hydrogen sulphide has been shown to induce apoptosis. However, a potential role of mitogen‐activated protein kinases (MAPKs) in this apoptotic pathway remains unknown. The present study examined the role of MAPKs in H2S‐induced apoptosis in mouse pancreatic acinar cells. Pancreatic acinar cells were treated with 10 μM NaHS (a donor of H2S) for 3 hrs. For the evaluation of the role of MAPKs, PD98059, SP600125 and SB203580 were used as MAPKs inhibitors for ERK1/2, JNK1/2 and p38 MAPK, respectively. We observed activation of ERK1/2, JNK1/2 and p38 when pancreatic acini were exposed to H2S. Moreover, H2S‐induced ERK1/2, JNK1/2 and p38 activation were blocked by pre‐treatment with their corresponding inhibitor in a dose‐dependent manner. H2S‐induced apoptosis led to an increase in caspase 3 activity and this activity was attenuated when caspase 3 inhibitor were used. Also, the cleavage of caspase 3 correlated with that of poly‐(ADP‐ribose)‐polymerase (PARP) cleavage. H2S treatment induced the release of cytochrome c, smac from mitochondria into the cytoplasm, translocation of Bax into mitochondria and decreased the protein level of Bcl‐2. Inhibition of ERK1/2 using PD98059 caused further enhancement of apoptosis as evidenced by annexin V staining, while SP600125 and SB203580 abrogated H2S‐induced apoptosis. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to H2S‐induced apoptosis.

Administration of exogenous fractalkine, a CX3C chemokine, is capable of modulating inflammatory response in cecal ligation and puncture-induced sepsis.

ABSTRACT Fractalkine (FTK) is a unique member of the CX3C chemokine family by acting through the CX3CR1 receptor. Membrane-bound FTK acts like an adhesion molecule, whereas soluble FTK (sFTK) acts as a classic chemokine ligand. Whether this chemokine plays a role in sepsis is still not clear. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis, we found that FTK levels were elevated in plasma 24 h after CLP. Reverse transcription-polymerase chain reaction results showed that FTK messenger RNA levels were upregulated, whereas CX3CR1 messenger RNA levels were downregulated in lungs after CLP procedure. To study the role of FTK in lung injury during sepsis, we injected exogenous sFTK into the mice before the CLP procedure. We found that plasma FTK levels were further elevated by sFTK. Mice that were injected with FTK had a lower myeloperoxidase activity in lungs compared with the CLP group. Furthermore, macrophage inflammatory protein 2, IL-1&bgr;, and IL-6 levels in lungs were reduced after the injection of FTK. Treatment with sFTK also attenuated lung morphological changes in histological sections. To find out whether sFTK had an effect on leukocyte rolling and adherence, intravital microscope was used. Results showed that sFTK significantly attenuated leukocyte adhesion but had little effect on leukocyte rolling in mesenteric microcirculation. Taken together, our findings suggest that FTK may be a novel chemokine that modulates neutrophil infiltration and chemokine and cytokine production during sepsis.