The findings of the present study demonstrate that intra-abdominally initiated sepsis has resulted in oxidative damage of the brain, heart, lung, liver and kidney within the first 25 h. During this period, treatment of the rats with NPW on three occasions reduced lipid peroxidation in the cardiac, pulmonary and cerebral tissues, while the depleted antioxidant GSH levels of the cerebral and cardiac tissues were replenished by NPW. NPW also suppressed the sepsis-related neutrophil recruitment to the hepatic and pulmonary tissues. Elevations in BUN and ALP, suggestive of renal and hepatic dysfunction, were not evident in the septic rats treated with NPW. Moreover, NPW treatment reduced the serum levels of CRP, corticosterone and the pro-inflammatory cytokine IL-6, while histopathologically verified tissue damage in all the studied tissues was ameliorated by NPW. Although survival rate of the rats was not significantly prolonged by NPW, most of these improvements in systemic and local inflammatory events were comparable with those reached by the etanercept and antibiotic combination, suggesting the therapeutic impact of NPW during the acute period of sepsis.
Observations in the septic shock patients have identified that CRP is expected to increase within the first 24–48 hours following the initial inflammatory response [29], while pro-calcitonin is elevated at nearly 2–4 hours after the onset of sepsis and shows a peak at 24–48 h [30]. In the rats induced with CLP, we observed that serum CRP level showed a significant elevation at the 25th h of sepsis, while the increases in pro-calcitonin and the pro-inflammatory IL-6 have not reached to statistical significance. On the other hand, sepsis induction has also resulted in high levels of corticosterone, indicating an enhancement in hypothalamo-pituitary-adrenal (HPA) axis activity. It is well documented that the initial activation of HPA axis during a bacterial infection involves the participation of cytokines and other inflammatory mediators [31], while homeostatic activation of the HPA axis and the actions of glucocorticoids are frequently impaired during the late periods of sepsis [32]. On the other hand, immunohistochemical studies have indicated that central administration of NPW, which is abundantly found in the nerve cell bodies and fibers in the hypothalamic areas associated with the HPA axis, was suggested to modulate the activation of HPA axis through its receptors in the hypothalamic nuclei [15, 33]. Furthermore, NPW was reported to directly stimulate corticosterone secretion by adrenocortical cells [34], while its intraperitoneal injection to healthy rats was also shown to increase plasma concentration of corticosterone [35]. However, in the present study, peripheral administration of NPW suppressed the synthesis of corticosterone that was increased by the upregulation of HPA axis activity via the inflammatory mediators of sepsis. Our findings suggest that peripherally administered NPW exerts an anti-inflammatory action, and thereby appears to control the release of inflammatory mediators that would exaggerate the HPA activity. During the progress of sepsis, serum levels of CRP, corticosterone and IL-6 were depressed in the septic rats treated with the antibiotic-anti-TNF-a combination, while analogous responses were obtained in NPW-treated septic rats, implicating that NPW may have comparable beneficial effects on the outcomes of polymicrobial sepsis.
Since sepsis-induced liver injury is associated with a high mortality, attenuation of liver damage and hepatic dysfunction is expected to improve the prognosis of septic patients [5, 36]. It was reported that hepatic dysfunction, which occurs as a consequence of hemodynamic instability and the injury of the hepatocytes and canaliculi, occurs within the first 1–2 h following CLP surgery in rodents [37]. CLP-induced hepatic injury was verified by our findings, demonstrating a histologically evident severe damage of the liver and a 2-fold increase in neutrophil infiltration. On the other hand, sepsis even due to non-respiratory causes can easily progress to acute respiratory distress syndrome, which is also associated with a great incidence of mortality in septic patients [38, 39]. Infiltration of neutrophils to the lung tissue is reported to be the major mechanism in inducing pulmonary capillary endothelial and alveolar epithelial injury [40, 41]. Our data support that acute pulmonary injury induced by intra-abdominal sepsis was accompanied by an enhanced infiltration of neutrophils to the lung tissue along with increased lipid peroxidation, while neutrophil infiltration or lipid peroxidation in the NPW-treated septic rats was not different than the control group, showing that the protective effect of NPW on the lung tissue involves the limitation of neutrophil-associated oxidative damage. In conjunction with that, NPW treatment ameliorated hepatic damage and prevented the elevation of ALP level, while facilitated neutrophil recruitment to the hepatic tissue with sepsis was totally abolished.
Clinical studies have shown that cerebral and cardiac injury may occur as early manifestations of sepsis-related organ damage and are also indicative of poor prognosis [42, 43]. It was reported that sepsis-induced cardiac dysfunction is caused mainly by elevated inflammation along with depleted energy and attenuated adrenergic signaling [44]. The pathophysiology of sepsis-related encephalopathy involves reduced cerebral blood flow, disrupted blood-brain barrier and the resultant neuronal degeneration due to adverse effects of inflammatory mediators, but leukocyte accumulation does not occur in the brain during sepsis [42]. Our current findings showed that sepsis-induced neuronal injury is not accompanied by any change in the accumulation of neutrophils, but involves a significant depletion of antioxidant GSH. Previous reports have also stated that oxidative damage in the target organs is caused by an imbalanced endogenous enzymatic activity [45, 46], suggesting that application of antioxidants could have a potential in the treatment of sepsis [11]. In the present study, alleviation of sepsis-induced neuronal injury by NPW treatment was accompanied by the replenished GSH stores, suggesting a stimulatory effect of NPW on the endogenous antioxidant capacity of the cerebral tissue. Similar to that has occurred in the cerebral tissue, sepsis has resulted in cardiac injury and an accompanying GSH depletion, which were not evident when the rats were treated with NPW. Thus, our results indicate that the protective effect of NPW treatment on both the cardiac and neuronal injury is directly related with the regulatory effect of NPW on tissue antioxidant content. There is substantial clinical evidence confirming that bacterial sepsis is responsible of nearly half of the cases with acute renal failure, and the coexisting renal failure is indicative of high mortality due to sepsis [47, 48]. In our CLP-induced sepsis model, oxidative injury was observed with increased lipid peroxidation and neutrophil infiltration in the renal tissue, as well as an elevated BUN level, showing the deterioration of the renal functions. However, NPW treatment was not equally efficient in reducing oxidative damage of the kidney as it was in alleviating sepsis- associated injury of the other target organs, but some amelioration in the histopathological changes and renal dysfunction was still evident.
Our data demonstrate that repetitive administration of NPW during the progress of sepsis, alleviated oxidative damage in all the studied target tissues. In order to evaluate the contribution of NFkB activity in the protective effects of NPW, we assessed the NFkB mRNA expressions in the target organs injured by intraabdominal sepsis induction. NFkB plays a critical role in the pathophysiology of sepsis and its activation leads to transcript stabilization and translation of the pro-inflammatory mediators [49]. Apart from its central role in the synthesis of cytokines and chemokines, NFkB also amplifies the expression, activation and survival of the inflammatory cells [50–52]. In both animal models and in human subjects with sepsis, NFkB activity was reported to be markedly increased in every target organ of the sepsis, while higher mortality rate and worse clinical outcome were associated with higher levels of NFkB activity, showing the profound impact of NFkB activation on the development of multiple organ dysfunction [53]. When NFkB-dependent genes were knockout or NFkB-signaling pathways were blocked, animals were found to be more resistant to the development of sepsis and its septic complications. Moreover, it was postulated that inhibition or modulation of NFkB could be beneficial in alleviating the inflammatory response and associated organ failure [54–57]. Our findings revealed that mRNA expression levels of NFkB were not significantly altered in the tissues insulted by sepsis, except for the renal tissue. On the other hand, treatment with NPW did not further change the depressed NFkB mRNA expression in the kidneys, while a profound inhibition of NFkB mRNA expression was evident in the lung tissue of NPW-treated septic rats, suggesting that the protective effect of NPW on sepsis-induced pulmonary injury could be mediated by suppressing oxidative stress via the inhibition of NFkB signaling pathway. Similarly, it was previously shown that inhibition of NFkB activation reduced the expressions of cytokines and adhesion molecules, neutrophil recruitment and abolished microvascular endothelial barrier permeability in the lungs of septic rats [58]. Although we have not observed any changes in the NFkB mRNA expression of the other sepsis-injured tissues, contribution of NFkB activity to the inflammatory cascades in these organs and the role of NPW during the earlier or delayed periods of sepsis cannot be ruled out.
Intrathecal administration of NPW was shown to produce analgesia against the inflammatory pain produced by formalin injection into the paw of the rats, but mechanically or thermally induced pain was not suppressed by NPW [20], implicating that NPW could be alleviating pain via its anti-inflammatory action. However, a possible anti-inflammatory effect of NPW against pain-provoking stimuli or its peripheral use was not thoroughly investigated. We have recently demonstrated that peripherally administered NPW exerts neuroprotective and antioxidant effects in rat pups with hypoxic-ischemic brain injury [21], and protects against stress-induced gastric ulcer in rats [22]. The present study further verifies the anti-inflammatory effects of NPW in multiple tissues affected by sepsis, and supports the notion that NPW or its agonists could be good candidates for the treatment of inflammation and for the relief of inflammatory pain.
Our study has some limitations. Since earlier studies have suggested that estrogens could protect the target tissues against sepsis-induced oxidative injury [59], use of only male rats is a limitation and neglects any varying actions that could occur in female rats. Studying in female rats at different stages of menstrual cycle could provide novel information about the interaction of NPW with hypothalamo-pituitary-gonadal axis. On the other hand, the protective effects of NPW could involve its blood pressure regulating effects [17–19], but in our experimental design we could not evaluate whether circulatory shock due to sepsis was affected by NPW. Moreover, we compared the effects of NPW with those of anti-TNF-a-antibiotic combination, but further experimental studies could be designed to investigate a possible synergistic effect.
In conclusion, our results revealed that NPW has effectively reduced oxidative stress in the target organs of rats subjected to acute CLP-sepsis. Although it seems unlikely that any single agent could reverse the severe and even lethal course of sepsis and provide a considerable clinical benefit, NPW and its agonists require further investigation for their novel therapeutic potential in alleviating organ dysfunction in various inflammatory diseases.