| The process of liver I/R injury is a cascade of inflammatory events involving multiple interconnected factors, including hepatic sinusoidal endothelial cell injury and disturbances of microvascular circulation, activation of Kupffer cells, production and release of reactive oxygen species and inflammatory mediators such as tumor necrosis factor (TNF)-a, interleukin (IL)-6, IL-1?, IL-18 and HMGB1.inflammasome are multiprotein cytoplasmic complexes that mediate the activation of inflammatory domain, leucine-rich repeat [LRR] domain, and pyrin domain [PYD]-containing protein-3). Caspase-1 cleaves pro-IL-lb to IL-lb and also activates IL-18 and IL-33. NALP3 is involved in the recognition of numerous exogenous and host ligands, including bacterial RNA, ATP, and uric acid crystals, and is also triggered by low concentrations of intracellular potassium (K+efflux) and increased levels of reactive oxygen species (ROS).A growing number of systemic inflammatory diseases, characterized by fever, anemia, and elevated levels of acute phase proteins, have been linked to excessive production and bioactivity of IL-1?. inflammasomes have been suggested to be involved in liver I/R injury, as the requisites for their activation, such as ROS, K+ efflux, and various damage-associated molecular pattern molecules (DAMPs), including HMGB1, DNA, and RNA, are released during hepatic ischemia. Furthermore, IL-1 family cytokines, including IL-lb and IL-18, which are secreted after inflammasome activation, have been implicated as promoters of liver I/R injury and some therapeutic strategies targeting IL-1b or IL-18 have proved to be effective in the protection of hepatocytes from I/R injury. In addition, there is evidence of a key role for IL-lb in the pathogenesis of I/R injury, as IL-lb knockout mice exhibit significant reduction of ischemic and reperfusion injury and, conversely, IL-1 receptor antagonist knockout mice exhibit increased I/R injury. However, whether blocking the NALP3 inflammasome signaling pathway could protect the liver from I/R injury is unknown.In the present study, we use a small hairpin RNA (shRNA) plasmid targeting NALP3 to examine the inhibitory effect of shRNA on the expression of NALP3 in vitro and in vivo, and explore the function and potential mechanisms of NALP3 in a murine model of liver I/R. Our studies demonstrate that gene silencing of NALP3 results in protection from inflammation and hepatocyte injury after liver I/R. In addition, we show that the protective effect of NALP3 silencing in liver I/R injury is caspase-1 dependent and is associated with reductions of inflammatory mediators (IL-1?, IL-18, TNF-a,IL-6 and HMGB1) and infiltration of macrophages and neutrophils. 1. NALP3 is involved in liver I/R injuryPrepare the model of liver ischemia and reperfusion injury with male C57/BL mice including sham group. (1) The changes of ALT and AST in serum and the morphological characters of liver in mice undergoing liver ischemia and reperfusion injury.We detected the levels of ALT and AST in serum in mice undergoing liver ischemia 1 hour and reperfusion 6 hours. At the same time, Ischemic lobes were fixed with 10% formalin. Samples were embedded in parafinn and cut into 4mm thick sections for hematoxylin-eosin (H&E) staining. The results show that the levels of ALT and AST were significant increased in model group than sham group(p<0.01) and the H&E staining show that there are a lot of hepatocytes denaturalization, apoptosis, necrosis and inflammatory infiltration in model mice. The above results indicated that the liver ischemia and reperfusion injury were successfully made. (2) The levels of IL-1?in serum at different time points after liver ischemia and reperfusion injury.We assessed over time the serum IL-1?level of sham mice and mice subjected to liver I/R by ELISA assay. Serum IL-lb protein expression increased after 1 hr of reperfusion, continued to increase, and was maximal at 6 hr of reperfusion. However, the IL-1b protein level after 24 hr of reperfusion were similar to that detected at 1 hr of reperfusion, and remained significantly greater than in sham controls. As the level of IL-lb peaked 6 hr after reperfusion, subsequent experiments were performed at this time. (3) The expression of NALP3 at different time points after liver ischemia and reperfusion injury.To explore whether NALP3 was involved in liver I/R injury, Western blot analysis was performed on liver from sham mice and mice subjected to liver I/R. NALP3 protein expression was significantly up regulated after 6 hr of reperfusion and lasted until 12 hr. (4) The level of ROS during mice liver ischemia and reperfusion injury.Isolate the NPC cells from mice underwent ischemia 1h and perfusion for 6 hours. We then examined the level of ROS in NPCs with DCF staining by FCSM. The results show that ROS of NPCs in liver I/R mice increased markedly compared with sham mice, indicating that NALP3 was activated during liver I/R. Thus, NALP3 signaling is required for the development of liver I/R injury. 2. Identification and construction of pNALP3shRNA vector (1) Scanning the effective sequenc of siRNAs for mouse NALP3 geneWe designed and selected three NALP3 siRNA sequences:siRNA1 (S1), siRNA2 (S2), and siRNA3 (S3), as well as a scrambled siRNA (SS) using the siRNA designer software, followed by transfection into CHO cells, which constitutively express NALP3 protein. siRNA3 (S3) was shown to inhibit NALP3 significantly, whereas the scrambled siRNA (SS) had no measurable effect on NALP3 expression. We determined that the siRNA3 sequence was the most effective sequence specifically targeting NALP3 protein. (2) Identification and construction of specific pNALP3shRNA plasmids.we successfully constructed the pNALP3shRNA and pshRNANC plasmids, using the siRNA3 interference sequence and scrambled siRNA sequence, respectively, and performed verification by restriction enzyme mapping and sequence analysis. The Kupffer cells were transfected with pNALP3shRNA or pshRNANC. The results indicated that the expression of NALP3 in Kupffer cells was significantly suppressed by pNALP3shRNA transfection compared with the mock treatment or pshRNANC transfection (p<0.05).In accordance with protein levels, pNALP3shRNA transfection reduced NALP3 mRNA levels compared with the pshRNANC and mock groups (p<0.05). In addition, the expression of NALPlb and NLRC4 were not changed after pNALP3shRNA treatment. (3) The changes of IL-1?level in kupffer cells after treatment of pNALP3shRNA.To further check the function of NALP3 silencing, we analyzed IL-lb release by Kupffer cells transfected with pNALP3shRNA or pshRNANC, as described in above. The release of IL-1 was significantly decreased in pNALP3shRNA-treated cells compared with mock and pshRNANC-treated cells. 3. The effects of NALP3silencing on mice ischemia and reperfusion injury and the mechanisms (1) The protective effect on liver ischemia and reperfusion injury with pNALP3shRNA pretreatment. 1) pNALP3shRNA inhibits NALP3 expression in vivoWe next checked the silencing of NALP3 after 6 hr of reperfusion in vivo. The results showed that hydrodynamic injection of 100 mg of pNALP3shRNA significantly inhibited the expression of NALP3 not only at the protein level, as determined by Western blotting (p<0.05) and immunohistochemical staining, but also at the mRNA level, as assessed by RT-PCR (p<0.05), whereas there was no significant difference in mice pretreated with pshRNANC and saline. NALP3 fluorescence in cells was measured by fluorescence-activated cell sorting (FACS), which showed significantly decreased fluorescence in both CD 146p liver sinusoidal endothelial cells (LSECs) and F4/80p Kupffer cellsin the pNALP3shRNA group compared with the pshRNANC and saline groups (p<0.05). 2) The effects of pNALP3shRNA on liver I/R injurySerum and liver samples were harvested 6,12, and 24 hr after reperfusion. The results show that,1 hr of warm hepatic ischemia followed by 6,12, and 24 hr of reperfusion significantly increased sALT and sAST levels in saline-and pshRNANC-pretreated mice subjected to I/R. Pretreatment with pNALP3shRNA resulted in significant protection from hepatic injury (p<0.01). Liver histology results confirmed the sALT and sAST estimation of liver damage at 6 hr of reperfusion. Large areas of necrosis were present in liver tissue from saline-and pshRNANC-pretreated mice whereas minimal damage was noted in samples from pNALP3shRNA-pretreated mice. (2) The mechanisms involved in the protective effect of silencing NALP3 on liver I/R injury1) The effects of pNALP3shRNA on IL-1?and IL-18 in liver I/R injurywe measured serum IL-1?and IL-18 in sham mice and mice pretreated with pNALP3shRNA, pshRNANC, or saline followed by 6 hr of reperfusion. Mice pretreated with pNALP3shRNA exhibited decreased circulating IL-lb and IL-18 levels compared with saline-or pshRNANC-pretreated mice (p<0.05), whereas there was no difference in mice pretreated with pshRNANC compared with saline-pretreated mice. The same with the cleaved caspase-1. pNALP3shRNA can also inhibitIL-1?release in kupffer cells in vitro.2) Gene silencing of NALP3 down regulates HMGBl,proinflammatory cytokines TNF-a and IL-6ELISA for TNF-a and IL-6 in serum and western blot analysis of HMGB1 expression were performed from mice that had been subjected to liver I/R and from sham mice. The levels of TNF-a and IL-6 in serum and HMGB1 protein expression were up regulated in the liver of saline-or pshRNANC-pretreated mice. In contrast, mice pretreated with pNALP3shRNA exhibited down regulated HMGB1 expression compared with pshRNANC-or saline-pretreated animals (p<0.05). After 1 hour of reperfusion, the NF-KB activity were tested by EMSA, the results show that pNALP3shRNA pretreatment significantly suppressed the activation of NF-kB (p<0.05). 3) The effect of pNALP3shRNA on apoptosis in liver I/R injuryThe mice were subjected to liver ischemia 1h and reperfusion 6 hours.The liver samples were harvested and fixed with 4% paraformaldehyde for paraffin sections. To detect the apoptosis of hepatocytes,we stained with TUNEL staining. The results shows that pretreatment of pNALP3shRNA can decrease the apoptosis of hepatocytes compared with pshRNANC and saline group (p<0.05).4) Knockdown of NALP3 decreases inflammatory cell infiltration in liver I/RWe investigated macrophage and neutrophil infiltration in liver I/R by immunohistochemical staining. The results showed that the infiltrated macrophages(F4/80) and neutrophils(Gr-1) were significantly decreased in pNALP3shRNA-pretreated mice than in pshRNANC-and saline-pretreated mice (p<0 .01). 4. conclusion:(1) NALP3 signaling is involved in mice liver ischemia and reperfusion injury.(2) Pretreatment of pNALP3shRNA can protect against liver ischemia and reperfusion injury in mice.(3) The mechanisms involved in this protect effects may includ inhibition of release of inflammatory cytokines such as IL-1??IL-18?TNF-?IL-6 and HMGB1,the down regulation of NF-KB activities, the decreased apoptosis of hepatocytes, and the reduced the inflammatory cell infiltration in liver. |
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