AIM:To explore the effects of CD38 on lysosome reformation and cholesterol efflux in macro-phages.METHODS:Bone marrow-derived macrophages from low-density lipoprotein(LDL)receptor knockout(LDLr-/-)mice were cultured as cell model.Live cell imaging system was applied to evaluate the effect of nicotinic acid adenine di-nucleotide phosphate(NAADP)on lysosome number.ELISA was conducted to measure NAADP level in macrophages.After the cells were treated with nicotinic acid(NA),RT-qPCR was conducted to detect CD38 mRNA expression,and Western blot was conducted to observe CD38 protein expression and phosphorylated transcription factor EB(TFEB)level.Laser scanning confocal microscopy was applied to evaluate the influence of CD38/NAADP signaling on lysosome number and cholesterol egression.RESULTS:NAADP remarkably increased lysosome number(P<0.05),and this effect was significantly inhibited by NAADP antagonist NED-19,Ca2+ chelator BAPTA,and calcineurin inhibitor CsA(P<0.05).CD38 markedly enhanced NAADP synthesis in macrophages(P<0.05).NAADP synthetic substrate NA prominently ele-vated the expression of CD38 mRNA and protein(P<0.05).NA significantly decreased the phosphorylated TFEB level;this effect was also attenuated by NED-19,BAPTA and CsA(P<0.05).Disrupting CD38/NAADP signaling pathway markedly inhibited NA-induced enhancement of lysosome number,lysosomal free cholesterol and cytosol cholesterol ester efflux in macrophages(P<0.05).NA-induced enhancement of lysosome number,lysosomal free cholesterol and cytosol cholesterol ester efflux abolished in LDLr/CD38 DKO macrophages(P<0.05),whereas these effects induced by NA were recovered after CD38 gene rescue.CONCLUSION:CD38 triggers lysosome reformation via TFEB and consequently pro-motes the efflux of lysosomal free cholesterol and cytosol cholesterol ester.

Objective To investigate the protective mechanism of Wenyang Fuyuan Prescription on nerve injury by improving brain iron metabolism in rats with cerebral ischemia-reperfusion injury(CIRI)based on ferroptosis.Methods A total of 72 SD rats were randomly divided into sham-operation group,CIRI model group,Wenyang Fuyuan Prescription group(18.0 g·kg-1,gavage),ferroptosis inducer group(100 mg·kg-1,intraperitoneal injection),Wenyang Fuyuan Prescription(18.0 g·kg-1,gavage)+ ferroptosis inducer group(intraperitoneal injection)and ferroptosis inhibitor group(5 mg·kg-1,intraperitoneal injection),12 rats in each group.All the procedures adopted in the sham group were the same as those in the model group.But nylon thread was inserted into the internal carotid artery at a depth of 9 mm and un-plugged middle cerebral artery.The rest of the groups were used to construct middle cerebral artery occlusion/reperfusion(MCAO/R)model by thread embolism method.Ferroptosis inducer(100 mg·kg-1)and ferroptosis inhibitor(5 mg·kg-1)were administered intraperitoneally to rats according to the grouping 24 hours before modeling.Wenyang Fuyuan Prescription(18.0 g·kg-1)was administered by gavage 2 hours after anesthesia and awakening.All intervention were given once daily for 7 consecutive days.The Longa scoring standard was used to evaluate the neurological deficit on 1,3,and 7 days after MCAO/R surgery,respectively.At the end of the treatment period,brain tissues were taken to observe the morphological changes of rat neurons in each group by hematoxylin eosin staining(HE).The ultrastructural changes of neuron mitochondria in each group were observed by transmission electron microscope.The biochemical kit was used to detect the content of iron ions(Fe2+)and reduced glutathione(GSH)in brain tissue.The protein and mRNA expressions of transferrin receptor 1(TFR1),iron regulatory protein 1(IRP1)and ferroportin(FPN)were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western Blot.Results① Compared with sham group,the neurological deficit scores of rats in model group increased at each time point(P＜0.01).HE staining showed neurons were sparse and disordered,the nuclei underwent pyknosis,and vacuoles appeared at the edges.Under electron microscopy,it was observed that the number of neuronal mitochondria decreased,the density of mitochondrial membranes increased,massive numbers of mitochondrial membranes ruptured and dissolved,and mitochondrial cristae disappeared.The content of Fe2+,both mRNA and protein expressions of TFR1 were significantly increased(P＜0.01),while GSH content,as well as expressions of mRNA and protein for IRP1 and FPN were significantly decreased(P＜0.05,P＜0.01).② Compared with the model group,the neurological deficit scores of rats in the Wenyang Fuyuan Prescription group decreased at various time points(P＜0.05).The number of neurons increased,their arrangement was relatively neat,the morphology of the nucleus is complete and clear,the mitochondrial structure of neurons was relatively complete,the mitochondrial membrane was relatively intact,and the mitochondrial cristae were clear.The content of Fe2+,both mRNA and protein expressions of TFR1 were decreased(P＜0.05,P＜0.01),while GSH content,as well as expressions of mRNA and protein for IRP1 and FPN increased(P＜0.05,P＜0.01).③ Compared with the Wenyang Fuyuan Prescription group,the neurological deficit scores of rats in ferroptosis inducer group and the Wenyang Fuyuan Prescription + ferroptosis inducer group increased at all time points(P＜0.05).Distribution of neurons was in disorder,the nucleus shrinked,and vacuoles appeared at the edges.The density of mitochondrial membranes increased,some ruptured and dissolved mitochondrial membranes were found.The number of mitochondria decreased and mitochondrial cristae disappeared.The content of Fe2+,both TFR1 mRNA and protein expression increased(P＜0.05,P＜0.01),while the content of GSH,as well as expressions of mRNA and protein for IRP1 and FPN decreased(P＜0.05,P＜0.01).However,there was no statistically significant difference in all observed indicators between the ferroptosis inhibitor group and the Wenyang Fuyuan Prescription group(P＞0.05).Conclusion Wenyang Fuyuan Prescription can improve the neurological function and pathological damage of CIRI rats.Its mechanism may be related to regulating the expression of IRP1 protein,improving the brain iron metabolism pathway,and inhibiting ferroptosis.