The innate immune sensor NLRP3 inflammasome overactivation is involved in the pathogenesis of ulcerative colitis. PGAM5 is a mitochondrial phosphatase involved in NLRP3 inflammasome activation in macrophages. However, the role of PGAM5 in ulcerative colitis and the mechanisms underlying PGAM5 regulating NLRP3 activity remain unknown. Here, we show that PGAM5 deficiency ameliorates dextran sodium sulfate (DSS)-induced colitis in mice via suppressing NLRP3 inflammasome activation. By combining APEX2-based proximity labeling focused on PGAM5 with quantitative proteomics, we identify NEK7 as the new binding partner of PGAM5 to promote NLRP3 inflammasome assembly and activation in a PGAM5 phosphatase activity-independent manner upon inflammasome induction. Interfering with PGAM5-NEK7 interaction by punicalagin inhibits the activation of the NLRP3 inflammasome in macrophages and ameliorates DSS-induced colitis in mice. Altogether, our data demonstrate the PGAM5-NEK7 interaction in macrophages for NLRP3 inflammasome activation and further provide a promising therapeutic strategy for ulcerative colitis by blocking the PGAM5-NEK7 interaction.

Studies have suggested that the nucleus accumbens (NAc) is implicated in the pathophysiology of major depression; however, the regulatory strategy that targets the NAc to achieve an exclusive and outstanding anti-depression benefit has not been elucidated. Here, we identified a specific reduction of cyclic adenosine monophosphate (cAMP) in the subset of dopamine D1 receptor medium spiny neurons (D1-MSNs) in the NAc that promoted stress susceptibility, while the stimulation of cAMP production in NAc D1-MSNs efficiently rescued depression-like behaviors. Ketamine treatment enhanced cAMP both in D1-MSNs and dopamine D2 receptor medium spiny neurons (D2-MSNs) of depressed mice, however, the rapid antidepressant effect of ketamine solely depended on elevating cAMP in NAc D1-MSNs. We discovered that a higher dose of crocin markedly increased cAMP in the NAc and consistently relieved depression 24 h after oral administration, but not a lower dose. The fast onset property of crocin was verified through multicenter studies. Moreover, crocin specifically targeted at D1-MSN cAMP signaling in the NAc to relieve depression and had no effect on D2-MSN. These findings characterize a new strategy to achieve an exclusive and outstanding anti-depression benefit by elevating cAMP in D1-MSNs in the NAc, and provide a potential rapid antidepressant drug candidate, crocin.

Objective To develop a high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method for simultaneous determination of five flavonoids in Ganmao'an granules(GMA).Methods Chromatographic separation was achieved on a Kromasil C18 Column(150 mm×4.6 mm,5 μm,100 ?),which was eluted with methanol(A)-0.1％formic acid(B)at the flow rate of 1.0 ml/min.The gradient condition was as follows:0-20 min,35％A,and 20-40 min,45％A.The column temperature was 25 ℃.Analytes were detected using a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source in the negative ion scanning.The multiple reaction monitoring mode was used for qualitative analysis.For each flavonoid,two precursor ion/product ion transitions were chosen:lutin m/z 609.1→300.1,hyperin and isoquercitrin m/z 463.0→300.1,quercetin m/z 301.0→151.0,luteolin m/z 285.0→132.9.Results Five flavonoids showed the good relationships within their own concentration ranges(correlation coefficient r>0.999 1),whose average recoveries were in the range of 100.63％-102.81％with RSDs of 0.67％-2.07％.The content results of rutin,hyperoside,isoquercetin,quercetin,and luteolin in 10 batches of GMA were 32.23-479.83,0.291-1.825,11.44-20.54,6.32-18.41,3.46-6.51 μg/g,respectively.Conclusion The results indicated that the developed method was sensitive,accurate and could provide excellent specificity for simultaneous determination of five flavonoids in GMA.