Akhil Hegde

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.

Preprotachykinin-A Gene Products Are Key Mediators of Lung Injury in Polymicrobial Sepsis

Preprotachykinin-A (PPT-A) gene products substance P and neurokinin-A have been shown to play an important role in neurogenic inflammation. To investigate the role of PPT-A gene products in lung injury in sepsis, polymicrobial sepsis was induced by cecal ligation and puncture in PPT-A gene-deficient mice (PPT-A−/−) and the wild-type control mice (PPT-A+/+). PPT-A gene deletion significantly protected against mortality, delayed the onset of lethality, and improved the long-term survival following cecal ligation and puncture-induced sepsis. PPT-A−/− mice also had significantly attenuated inflammation and damage in the lungs. The data suggest that deletion of the PPT-A gene may have contributed to the disruption in recruitment of inflammatory cells resulting in protection against tissue damage, as in these mice the sepsis-associated increase in chemokine levels is significantly attenuated.

Treatment with antileukinate, a CXCR2 chemokine receptor antagonist, protects mice against acute pancreatitis and associated lung injury.

Acute pancreatitis is a common, and as yet incurable, clinical condition, the incidence of which has been increasing over recent years. Chemokines are believed to play a key role in the pathogenesis of acute pancreatitis. We have earlier shown that treatment with a neutralizing antibody against CINC, a CXC chemokine, protects rats against acute pancreatitis-associated lung injury. The hexapeptide antileukinate (Ac-RRWWCR-NH2) is a potent inhibitor of binding of CXC chemokines to the receptors (CXCR2). This study aims to evaluate the effect of treatment with antileukinate on acute pancreatitis and the associated lung injury in mice. Acute pancreatitis was induced in adult male Swiss mice by hourly intra-peritoneal injections of caerulein (50 microg/kg/h) for 10 h. Antileukinate (52.63 mg/kg, s.c.) was administered to mice either 30 min before or 1 h after starting caerulein injections. Severity of acute pancreatitis was determined by measuring plasma amylase, pancreatic water content, pancreatic myeloperoxidase (MPO) activity, pancreatic macrophage inflammatory protein-2 (MIP-2) levels and histological examination of sections of pancreas. A rise in lung MPO activity and histological evidence of lung injury in lung sections was used as criteria for pancreatitis-associated lung injury. Treatment with antileukinate protected mice against acute pancreatitis and associated lung injury, showing thereby that anti-chemokine therapy may be of value in this condition.

Neurokinin-1 receptor antagonist treatment protects mice against lung injury in polymicrobial sepsis

Earlier work from our laboratory has suggested a role for the neuropeptide substance P (SP) in inducing lung injury in sepsis. In that study, mice lacking the preprotachykinin‐A gene, which encodes for SP, were protected against lung injury in sepsis. To further substantiate the role of SP in sepsis and to study its mchanism, we have evaluated the effect of SR140333, a SP receptor antagonist, on lung injury in sepsis, which was induced in male Swiss mice by cecal ligation and puncture (CLP). Sham‐operated animals received the same surgical procedure, except CLP. Vehicle or SR140333 (1 mg/kg, s.c.) was administered to CLP mice 30 min before or 1 h after the CLP. Eight hours after surgery, lung tissue was collected and analyzed for myeloperoxidase (MPO) activity, chemokines, cytokines, and adhesion molecules. The CLP procedure alone caused a significant increase in the lung levels of MIP‐2, MCP‐1, IL‐1β, IL‐6, ICAM‐1, E‐ and P‐selectin, and MPO activity when compared with sham‐operated mice. SR140333 injected 30 min before or 1 h after CLP significantly attenuated the increased lung MPO activity and levels of MIP‐2, MCP‐1, IL‐1β, IL‐6, ICAM‐1, and E‐ and P‐selectin compared with CLP‐operated mice injected with the vehicle. Histological evaluation of the lung sections further supported the beneficial effect of SR140333 on lung inflammation. Therefore, SP receptor antagonism can be a potential therapeutic target in polymicrobial sepsis, and this effect is brought about via reduction in leukocyte recruitment.

The effect of CSE gene deletion in caerulein-induced acute pancreatitis in the mouse

Hydrogen sulfide (H2S) has been reported to be involved in the signaling of the inflammatory response; however, there are differing views as to whether it is pro- or anti-inflammatory. In this study, we sought to determine whether endogenously synthesized H2S via cystathionine-γ-lyase (CSE) plays a pro- or anti-inflammatory role in caerulein-induced pancreatitis. To investigate this, we used mice genetically deficient in CSE to elucidate the function of CSE in caerulein-induced acute pancreatitis. We compared the inflammatory response and tissue damage of wild-type (WT) and CSE knockout (KO) mice following 10 hourly administrations of 50 μg/kg caerulein or saline control. From this, we found that the CSE KO mice showed significantly less local pancreatic damage as well as acute pancreatitis-associated lung injury compared with the WT mice. There were also lower levels of pancreatic eicosanoid and cytokines, as well as reduced acinar cell NF-κB activation in the CSE KO mice compared with WT mice. Additionally, in WT mice, there was a greater level of pancreatic CSE expression and sulfide-synthesizing activity in caerulein-induced pancreatitis compared with the saline control. When comparing the two saline-treated control groups, we noted that the CSE KO mice showed significantly less pancreatic H2S-synthesizing activity relative to the WT mice. These results indicate that endogenous H2S generated by CSE plays a key proinflammatory role via NF-κB activation in caerulein-induced pancreatitis, and its genetic deletion affords significant protection against acute pancreatitis and associated lung injury.

Role of preprotachykinin-A gene products on multiple organ injury in LPS-induced endotoxemia

Endotoxemia is a life‐threatening, inflammatory condition that involves multiple organ injury and dysfunction. Preprotachykinin‐A (PPT‐A) gene products, substance P (SP), and neurokinin‐A have been shown to play an important role in neurogenic inflammation. To investigate the role of PPT‐A gene products on multiple organ injury in LPS‐induced endotoxemia, endotoxemia was induced by LPS administration (10 mg/kg, i.p.) in PPT‐A gene‐deficient mice (PPTA−/−) and the wild‐type (WT) control mice (PPT‐A+/+). I.p. administration of LPS to WT mice caused a significant increase in circulating levels of SP as well as in liver, lung, and kidney. PPT‐A gene deletion significantly protected against liver, pulmonary, and renal injury following LPS‐induced endotoxemia, as evidenced by tissue myeloperoxidase activities, plasma alanine aminotransferase, aspartate aminotransferase levels, and histological examination. Furthermore, PPT‐A−/− mice had significantly attenuated chemokines, proinflammatory cytokines, and adhesion molecule levels in the liver, lung, and kidney. These results show that PPT‐A gene products are critical proinflammatory mediators in endotoxemia and the associated multiple organ injury. In addition, the data suggest that deletion of the PPT‐A gene protected mice against organ damage in endotoxemia by disruption in neutrophil recruitment.

Current trends in modern pharmaceutical analysis for drug discovery

Traditionally, pharmaceutical analysis referred to the chemical analysis of drug molecules. However, over the years, modern pharmaceutical analysis has evolved beyond this to encompass combination techniques, high-throughput technologies, chemometrics, microdosing studies, miniaturization and nanotechnology. These analytical advances are now being employed in all stages of drug discovery and the focus of this review will be on how these technologies are being employed within this process. With new, improved and evolving technologies, as well as new applications for existing technology, the search for new drugs for the prevention and treatment of human diseases continues.

Hydrogen sulfide in inflammation: friend or foe?

Hydrogen sulfide (H(2)S), the gaseous mediator produced by various cells in our body, was recently discovered to play a major role in human physiology despite its toxic nature known for centuries. In addition to its pathophysiological relevance in cardiovascular and neuronal disorders, there is considerable interest in the significance of H(2)S in inflammation. A number of preclinical studies in our laboratory as well as by others, using H(2)S donors and inhibitors of its endogenous synthesis, have provided evidence for both pro- and anti-inflammatory character of H(2)S. But so far, there is a significant lack of support from relevant clinical studies. One of the major contentious issues being variable dose and sampling time, controversies exist on the precise friend or foe nature of this gaseous transmitter. However, it is well accepted that once a clearer picture of the whole story of H(2)S in inflammation emerges, potential for therapeutic manipulations in this field are immense. This review focuses on the intriguing effects of H(2)S in some of the inflammatory conditions such as acute pancreatitis, sepsis, burn injuries and local inflammation of the joints. Active research projects have been undertaken to elucidate the mechanisms of action of H(2)S in inflammation, including neurogenic inflammation and interaction with other biological mediators and pathways. The early and fragmentary evidence obtained holds promise for a successful drug intervention for these inflammatory diseases.

Effects of S-Propargyl-Cysteine (SPRC) in Caerulein-Induced Acute Pancreatitis in Mice

Hydrogen sulfide (H2S), a novel gaseous messenger, is synthesized endogenously from L-cysteine by two pyridoxal-5′-phosphate-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). S-propargyl-cysteine (SPRC) is a slow H2S releasing drug that provides cysteine, a substrate of CSE. The present study was aimed to investigate the effects of SPRC in an in vivo model of acute pancreatitis (AP) in mice. AP was induced in mice by hourly caerulein injections (50 µg/kg) for 10 hours. Mice were treated with SPRC (10 mg/kg) or vehicle (distilled water). SPRC was administered either 12 h before or 3 h before the induction of pancreatitis. Mice were sacrificed 1 h after the last caerulein injection. Blood, pancreas and lung tissues were collected and processed to measure the plasma amylase, plasma H2S, myeloperoxidase (MPO) activities and cytokine levels in pancreas and lung. The results revealed that significant reduction of inflammation, both in pancreas and lung was associated with SPRC given 3 h prior to the induction of AP. Furthermore, the beneficial effects of SPRC were associated with reduction of pancreatic and pulmonary pro-inflammatory cytokines and increase of anti-inflammatory cytokine. SPRC administered 12 h before AP induction did not cause significant improvement in pancreatic and lung inflammation. Plasma H2S concentration showed significant difference in H2S levels between control, vehicle and SPRC (administered 3 h before AP) treatment groups. In conclusion, these data provide evidence for protective effects of SPRC in AP possibly by virtue of its slow release of endogenous H2S.

Neurokinin-1 Receptor Antagonist Treatment in Polymicrobial Sepsis: Molecular Insights

Neurokinin-1 receptor blocking has been shown to be beneficial against lung injury in polymicrobial sepsis. In this paper, we evaluated the possible mediators and the mechanism involved. Mice were subjected to cecal ligation and puncture (CLP-) induced sepsis or sham surgery. Vehicle or SR140333 [1 mg/kg; subcutaneous (s.c.)] was administered to septic mice either 30 min before or 1 h after the surgery. Lung tissue was collected 8 h after surgery and further analyzed. CLP alone caused a significant increase in the activation of the transcription factors, protein kinase C-α, extracellular signal regulated kinases, neurokinin receptors, and substance P levels in lung when compared to sham-operated mice. SR140333 injected pre- and post surgery significantly attenuated the activation of transcription factors and protein kinase C-α and the plasma levels of substance P compared to CLP-operated mice injected with the vehicle. In addition, GR159897 (0.12 mg/kg; s.c.), a neurokinin-2 receptor antagonist, failed to show beneficial effects. We conclude that substance P acting via neurokinin-1 receptor in sepsis initiated signaling cascade mediated mainly by protein kinase C-α, led to NF-κB and activator protein-1 activation, and further modulated proinflammatory mediators.