Effect And Mechanism Of Extracellular Acidosis On NLRP1 Inflammasome Activation In Primary Culture Cortical Neurons

Inflammasome,a multiprotein complex in the cytoplasm scaffolded by intracellular pattern recognition receptors(PRRs), is an important component of the innate immune system. The NLRP1(NOD-like receptor(NLR) family, pyrin domain containing 1) inflammasome, which comprised caspase-1, the adaptor protein apoptosis-associated speck-like protein containing a caspase-activating recruitment domain protein(ASC), and the sensor NLRP1, was the first member of the NLR family to be discovered. It expressed in neurons and involved in p Hysiological and pathological processes of many neuronal diseases. Acidosis is a pathological condition in which acid-base balance is disturbed in the direction of excess acidity in the body fluid. It is a common feature in many central nervous system(CNS) diseases. However, the effect of acidosis on NLRP1 inflammasome remains unclear.Objective To investigate the effect and mechanism of exetracellular acidosis on NLRP1 inflammasome activation and determine the role of NLRP1 inflammasome in acidosis-induced neurons injury in primary culture cortical neurons.Methods Western blot was used to detect the expression of NLRP1、caspase-1、ASC、IL-1β、IL-18、caspase-3、Bax and Bcl2 under acidosis and treatment with drugs in primary culture cortical neurons. Whole cell patch clamp recording was used to observe the effect of exetracellular acidosis on BK currents in cortical neurons. Co-expression of ASICs and BK channel was investigated by immunofluorescent double. Ion-selective microelectrode method was used to detect [K]i under acidosis and treatment with drugs in primary culture cortical neurons. The cell viability and LDH release were detected by MTT and LDH kit.Result 1. Compared with control group(p H 7.4), exetracellular acidosis(p H 7.0, p H 6.5, p H 6.0) increase the expression of NLRP1、caspase-1、ASC、IL-1βand IL-18, suggesting extracellular acidosis activates NLRP1 inflammasome in primary culture cortical neurons.2. Extracellular acidosis increases BK currents and decreases [K]i in cortical neurons. IBTX, a BK channel inhibitor, attenuated acidosis induced decrease in [K]i3. ASICs and BK channel are co-expressed in cortical neurons. ASICs inhibitor amiloride and specific ASIC1 a inhibitor Pc TX1 inhibited acidosis induced increase of BK currents. Pc TX1 also attenuated acidosis induced decrease in [K]i4. BK channel inhibitor IBTX attenuated acidosis(p H 6.5) induced increase in expression of NLRP1, caspase-1, ASC, IL-1β and IL-18, indicating BK channel involve in NLRP1 inflammasome activation induced by extracellular acidosis.5. Specific ASIC1 a inhibitor Pc TX1 attenuated acidosis(p H 6.5) induced increase in expression of NLRP1, caspase-1, ASC, IL-1β and IL-18, indicating ASICs involve in NLRP1 inflammasome activation induced by extracellular acidosis.6. Extracellular acidosis(p H 6.5) decrease cell viability and the expression of Bcl2, and increase LDH release and the expression of caspase-3 and Bax in primary culture cortical neurons. Inhibition of NLRP1 inflammasome activation by NLRP1 Ab(1:100)blocked acidosis affect on cortical neurons, suggesting NLRP1 inflammasome involve in neurons injury induced by extracellular acidosis.Conclusion ASICs-BK channel contributes to NLRP1 inflammasome activation under extracellular acidosis, which involve in neurons injury induced by extracellular acidosis.