Inhibitory Effects And Mechanisms Of Baicalin On Monosodium Urate Induced Pyroptosis Of Human Kidney-2 Cells

Hyperuricemic nephropathy(HN)is a serious complication of hyperuricemia,and one of the main pathological characteristics is the deposition of monosodium urate(MSU)in the kidney,which leads to severe renal inflammation.At present,the drugs for HN treatment are mainly to reduce the level of uric acid or anti-inflammatory therapy,but all have many toxic side effects.Pyroptosis is a type of programmed cell death(PCD),characterized by changes in cell membrane permeability and release of inflammatory cytokines into the extracellular cell,followed by more severe inflammatory reactions.Baicalin(BA)is a flavonoid compound extracted from the root of the traditional Chinese medicine scutellaria baicalensis.Previous studies have shown that BA can reduce uric acid level through significant anti-xanthine oxidase activity,BA also has a wide range of anti-inflammatory and anti-oxidant stress activities.In this study,the MSU-induced injury model of human renal tubular epithelial cells(HK-2)was established.Morphological observation,biochemical index detection,western blot,RT-q PCR,molecular docking,molecular dynamics simulation,protein inhibitors and other techniques were used to investigate the effect of BA on MSU-induced pyroptosis of HK-2 cells and its mechanism.(1)Use BA(20,40,80 μM)to interfere with the effects of MSU(2 mg/m L)on HK-2 cell injury for 12 h.The extracellular LDH level was detected and the cells membrane was observed by microscopic observation.Western blotting and RT-q PCR were used to detect the level of pyroptosis related genes and proteins including NLRP3、ASC、Caspase-1、GSDMD、IL-1β.The results showed that MSU-induced HK-2 cells suffered severe membrane damage.The gene and protein levels of classical pyroptosis pathway were significantly increased.BA(40,80 μM)significantly inhibited the above MSU-induced cell damage,and down-regulated the classical pyroptosis pathway.(2)Molecular docking and molecular dynamics simulation techniques were used to explore the interaction between BA and proteins related to the classical pyroptosis pathway including NLRP3、ASC、Caspase-1、GSDMD.The results indicated that BA played a anti-pyroptosis effects through inhibiting the assembly of inflammatory bodies and the oligomerization of gasdermin-D.(3)Use BA(80 μM),Panx-1 inhibitor probenecid(100 μM)and P2X7 inhibitor bright blue G(5 μM)on the purinergic pathway to interfere with the effects of MSU(2 mg/m L)on HK-2 cell injury for 12 h.The extracellular LDH level was detected and the cells membrane was observed by microscopic observation.The results showed that MSU up-regulated the gene and protein expressions of Panx-1 and P2X7,and blocking the purinergic pathway with probenecid and bright blue G significantly down-regulated the gene and protein levels of NLRP3,ASC,Caspase-1,GSDMD,IL-1β and IL-18 in the classical pyrogenic pathway.BA(80 μM)significantly inhibited both purinergic and pyroptosis pathways.In conclusion,MSU activates the Panx-1/P2X7 purinergic signaling pathway in HK-2 cells and the NLRP3 inflammasome mediated classical pyroptosis pathway,which induces severe pyroptosis of renal cells in the high uric acid environment.BA can down-regulate the purinergic pathway and classical pyroptosis pathway activated by MSU,and exert anti-HN effect by reducing extracellular injury signal transduction and renal cell pyroptosis,which has considerable potential in treating HN.