The Roles Of Kir6.2/K-ATP Channel In LPS-induced Mouse Liver Injury

It is known that liver is an important metabolic organ of the body in mammals and humans, charged with nutrition metabolism, detoxification, immunologic defence and other vital physiological functions. Liver diseases caused by hepatocytes injury with high morbidity and mortality characteristics, becomes a serious threat to human health.Hepatocyte apoptosis, increased release of proinflammatory cytokines, reactive oxygen species (ROS) accumulation and endoplasmic reticulum stress and other mechanisms are involved in the development and progression of liver disease. Wherein, the inflammation may be the most important factors leading to liver damage. Recent studies suggest that inflammasome-mediated inflammatory responses play an important role in liver injury. Inflammasome is an intracellular multi-molecular complex composed of pattern recognition receptors NLRs, apoptosis-associated speck-like protein containing a CARD domain and procaspase-1.Damage -associated molecular pattern (Damage-associated molecular patterns, DAMPs), and pathogen-associated molecular patterns (Pathogen-associated molecular patterns, PAMPs)activate the inflammasome, promoting maturation and secretion of inflammatory cytokines IL-1β and IL-18. Currently, as is known,inflammasome activation is involved in the development of autoimmune diseases, atherosclerosis, type 2 diabetes, gout and other diseases. In addition, it is reportedthat NLRP3 is highly expressed in the hepatic parenchymal cells and non-parenchymal cells. NLRP3 or caspase-1 knockout or IL-1 receptor antagonist (IL-1Ra) may alleviate liver damage, which shows that NLRP3 inflammasome activation is linked to inflammation induced hepatocytes damage. Therefore, to find the molecular target and ideal drugs for regulation of liver inflammation has great significance to reduce liver damage and effecient treatment of liver diseases.ATP-sensitive potassium (K-ATP)channel is non-voltage-dependent ATP-sensitive potassium ion channels coupling cell metabolism to electrical activity in the body, as an energy sensor to regulate metabolic activity in the body. K-ATP channels regulate insulin secretion, hepatic gluconeogenesis, relaxing vascular smooth muscle to regulate blood pressure, as well as other important physiological functions. Itis documented, Kir6.2/K-ATP channel subunit is highly expressed in the liver tissue, opening K-ATP channels can upregulate heme oxygenase-1, thus relieving ischemia-reperfusion injury in rat liver transplantation, indicating that K-ATP channels may play an important role in liver injury. In recent years, our laboratory study found that opening K-ATP channel can inhibit production of ROS and inflammatory factors iNOS, TNF-a in microglia and endothelial cells, as a result, reducing the central and peripheral inflammation, suggesting that K-ATP channel has protective effect of inhibition of inflammation and injury reduction. So, whether Kir6.2/K-ATP channel, highly expressed in the liver tissue has protective effect on liver damage caused by inflammation, pending further study.Firstly, Kir6.2-/- mice is applied to establish the model of LPS-induced liver injury,investigating the correlation of Kir6.2/K-ATP channels and LPS-induced liver injury in mice and its mechanism behind them; primary Kupffer cells were isolated and cultured to found inflammatory cell injury model, to observe protective effect of Kir6.2/K-ATP channel openers Iptakalim (IPT) on LPS-induced liver injury and clarifying that the regulation role of Kir6.2/K-ATP channels in NLRP3 inflammasome activation. Ultimately, our study reveals Kir6.2/K-ATP channels are involved in the pathological process of LPS-induced liver injury, providing new ideas and strategies for clinical therapeutics of inflammation-related liver disease.AIM:Kir6.2 knockout mice were utilized to investigate the effect and the mechanism of Kir6.2/K-ATP channel on LPS-induced liver injury model.METHODS:1. Kir6.2+/+ and Kir6.2-/- mice were injected with LPS for 6 h to establish liver injury model.2.liver tissue is stained By Haematoxylin and eosin, and serum alanine aminotransferase (Alanine aminotransferase, ALT) levels were determined, observing the effect of Kir6.2 knockout on LPS-induced liver injury.3. Enzyme-linked immunosorbent assay (ELISA) was taken to detect the secretion levels of proinflammatory cytokines IL-1β, IL-18, TNF-a in serum afer LPS stimulation.4. Western blotting was employed to observe the effects of Kir6.2 knockout on LPS-induced NLRP3 inflammasome activation-related indicators of liver tissue in mice:the nuclear transcription factor NF-kB (nuclear factor kappa B) subunit p65, nucleotide-binding oligomerization domain (NOD)-like receptor 3 (NLRP3), caspase-1 (caspase-1), interleukin-1(3 (IL-1β); endoplasmic reticulum stress-related indicators:glucose-regulated protein 78 (Grp78), C/EBP homologous protein (CHOP), semi-cysteine aspartic protease-12 (caspase-12); autophagy-related indicators:microtubule-associated protein 1 light chain 3 (LC3II), p62 expression.5. cultured primary hepatocytes and Kupffer cells were seperated from Kir6.2+/+ and Kir6.2-/- mice, and were exposed to LPS (100ng/mL) and ATP (5 mM). Western blotting was applied to observe the effects of Kir6.2 knockout on LPS and ATP-induced NLRP3 inflammasome activation in primary Kupffer cells. ELISA was utilized to determine the levels of pro-inflammatory cytokines IL-1β, and IL-18 secreted from Kupffer cellsRESULTS:1. Kir6.2knockout aggravated liver injury in mice after LPS challengeAfter LPS (15mg/kg.ip) treatment,1) Kir6.2 protein expression was significantly decreased in the liver of Kir6.2+/+ mice (P<0.05).2) The level of serum ALT in Kir6.2-/- mice was significantly higher than WT mice (P<0.05).3)There is a significantly increased neutrophils and lymphocytes infiltration into liver in Kir6.2-/-mice than in Kir6.2+/+ mice.2. Kir6.2knockoutpromoted CHOP and caspase-12 mediated hepatocytes injury.Under basal condition, expression of CHOP, caspase-12 in mice of two genotypes was no significant difference (P> 0.05), expression of GRP78 in the liver of Kir6.2-/- mice was significantly higher than in WT mice (P<0.05); Kir6.2 knockout significantly enhanced LPS-induced GRP78, CHOP, caspase-12 expression in liver (P<0.05); Kir6.2 knockout significantly promote upregulation of LC3Ⅱ expression (P<0.05), markedly reduced the expression of p62 in liver (P<0.05).3. Kir6.2knockout accelerated NF-κB signaling pathway activation and enhanced the secretion of pro-inflammatory cytokinesAfter LPS (15mg/kg, i.p.) stimulation,1) The release level of inflammatory cytokines IL-1β (P<0.05), IL-18 (P<0.01), TNF-α (P<0.05) in Kir6.2-/- mice were significantly increased compared to Kir6.2+/+ mice; 2) an increase of nuclear translocation of p65 subunit in liver is significantly higher than Kir6.2+/+ mice (P<0.05).4. Kir6.2knockoutreinforced NLRP3 inflammasome significantly activation in the liver tissueAfter LPS (15mg/kg, i.p.) challenge,1) Expression of NLRP3, caspase-1 activation and expression of the mature form of IL-1β were significantly elevated in the liver of Kir6.2-/- mice than Kir6.2+/+ mice, namely, Kir6.2 knockout significantly promote the activation of NLRP3 inflammasome.5. Kir6.2knockout augmented NLRP3 inflammasome significantly activation and pro-inflammatory cytokines secretion in Primary Kupffer cells1) Primary hepatocytes and primary Kupffer cells were cultured, and IL-1β levels secreted from Kupffer cells were significantly higher than hepatocytes(P<0.01); after the treatment in primary Kupffer cells with LPS (100 ng/ml) and ATP (5mM),2) NLRP3 inflammasome was significantly increased in Kupffer cells in Kir6.2-/- mice compared with Kir6.2+/+ mice (P<0.05); 3)The levels of pro-inflammatory cytokines IL-1β (P<0.01), IL-18 (P<0.05) secreted from Kupffer cells in Kir6.2-/- mice were significantly augmented compared to Kir6.2+/+ mice.6.K-ATP channel opener IPT repressed LPS-induced inflammatory response in liver and alleviated liver injuryIPT significantly inhibited LPS-induced lymphocytes and neutrophils infiltration into the liver of Kir6.2+/+ mice and elevation of serum ALT levels (P<0.05), however, IPT cannot decrease LPS-induced exacerbations infiltration of lymphocytes and liver addicted neutrophils and increasement of serum ALT levels in the Kir6.2-/- mice (P> 0.05); IPT significantly restrained the activation of NLRP3 inflammasome in the liver of mice (P<0.05) and secretion of pro-inflammatory cytokines IL-1β,IL-18 (P<0.01); IPT significantly suppressed NLRP3 inflammasome activation in Kupffer cells (P<0.01).CONCLUSION:Kir6.2 knockout aggravated LPS-induced liver injury via augmenting NLRP3 inflammasome activation-mediated inflammatory response; K-ATP channelopener IPT alleviated LPS-induced mouse liver injury by repressing NLRP3 inflammmasome-mediated inflammatory response. Therefore, Kir6.2/K-ATP channel might be a potential target to the prevention and therapy for liver injury; K-ATP channelopener may be a potential in relieving liver injury.The major contributions of the present study lie in:1. The results revealthe relevance of Kir6.2/K-ATP channel to liver injury by establishing LPS-induced liver injury in Kir6.2-/- and Kir6.2+/+ mice.2. Our study clarifies that Kir6.2/K-ATP channel relievinghepatocytes injury caused by inflammation via modulating the NLRP3 inflammasome activation in Kupffer cells, providing the empirical evidence for the therapy of inflammatory-related diseases through targeting to Kir6.2/K-ATP channel.